KR102441231B1 - Method for producing polypeptide heteromultimer - Google Patents

Method for producing polypeptide heteromultimer Download PDF

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KR102441231B1
KR102441231B1 KR1020167010601A KR20167010601A KR102441231B1 KR 102441231 B1 KR102441231 B1 KR 102441231B1 KR 1020167010601 A KR1020167010601 A KR 1020167010601A KR 20167010601 A KR20167010601 A KR 20167010601A KR 102441231 B1 KR102441231 B1 KR 102441231B1
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도모유키 이가와
히토시 가타다
후타 미모토
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Abstract

본 발명은 전하 반발을 이용하여 Fc영역의 해리의 촉진 및/또는 회합의 제어를 달성하는, Fc영역의 계면을 형성하는 아미노산에 있어서의 개변 및/또는 중쇄 CH3영역의 안정성을 불안정하게 하는 개변을 중쇄 CH3영역에 도입한 복수 종류의 폴리펩티드의 호모체를 환원 조건하에서 인큐베이트함으로써 이종 다량체를 효율적이고 또한 안정하게 제조하는 방법을 제공한다.The present invention uses charge repulsion to promote dissociation of the Fc region and/or to control association, alterations in amino acids forming the interface of the Fc region and/or modifications that destabilize the stability of the heavy chain CH3 region Provided is a method for efficiently and stably producing a heteromultimer by incubating homomers of a plurality of types of polypeptides introduced into the heavy chain CH3 region under reducing conditions.

Description

폴리펩티드 이종 다량체의 제조방법{Method for producing polypeptide heteromultimer}Method for producing a polypeptide heteromultimer {Method for producing polypeptide heteromultimer}

본 발명은 폴리펩티드 이종 다량체의 제조방법 및 폴리펩티드의 헤테로화가 촉진되도록 Fc영역의 아미노산이 개변된 폴리펩티드 이종 다량체 등에 관한 것이다.The present invention relates to a method for producing a polypeptide heteromultimer, and to a polypeptide heteromultimer in which the amino acid of the Fc region is modified to promote heterogeneity of the polypeptide.

항체는 혈중에서의 안정성이 높고 부작용이 적은 것으로부터 의약품으로서 주목되고 있다(비특허문헌 1, 비특허문헌 2). 그 중에는 2종류의 항원 또는 에피토프를 동시에 인식할 수 있는 이중특이성 항체가 있고, 이들은 표적 특이성의 높음과 복수의 경로를 동시에 저해하는 기능이 기개된다(비특허문헌 3). 예를 들면 이미 시판되고 있는 카투막소맵(Catumaxomab)은 상피세포 접착인자인 EpCAM과, T세포에 발현하고 있는 CD3에 결합하는 이중특이성 항체로, 악성 복수의 치료제로서 사용되고 있다. Antibodies are attracting attention as pharmaceuticals because of their high stability in blood and few side effects (Non-Patent Document 1, Non-Patent Document 2). Among them, there are bispecific antibodies capable of simultaneously recognizing two types of antigens or epitopes, and these have high target specificity and a function of simultaneously inhibiting multiple pathways (Non-Patent Document 3). For example, Catumaxomab, which is already commercially available, is a bispecific antibody that binds to EpCAM, an epithelial cell adhesion factor, and CD3, which is expressed on T cells, and is used as a treatment for malignant ascites.

IgG형 이중특이성 항체의 제조는 목적으로 하는 이중특이성 항체가 얻어지는 효율의 낮음과, 정제의 곤란함으로부터 난이도가 높으나, 효율적 생산에 관한 지견이 몇 가지 보고되어 있다(비특허문헌 3). 예를 들면 2종류의 가변영역을 갖는 IgG를 구성하는 H쇄 및 L쇄의 유전자, 합계 4종의 유전자를 세포에 도입하고 공발현에 의해 분비하는 경우, 2종류의 H쇄의 공유 결합이나 H쇄와 L쇄의 비공유 결합은 랜덤으로 일어나기 때문에, 목적의 이중특이성 항체의 비율은 매우 적어져 생산 효율이 현저히 저하된다. 이를 해결하는 수단으로서, IgG H쇄의 CH3영역에 아미노산 치환을 행함으로써 H쇄에 대해서 이종인 조합의 IgG를 우선적으로 분비할 수 있는 것이 보고되어 있다(특허문헌 1 및 비특허문헌 4 및 5). 본 수법은 한쪽 H쇄의 CH3영역에 존재하는 아미노산 측쇄를 보다 큰 측쇄(knob;돌기)로 치환하고, 다른 한쪽 H쇄의 CH3영역에 존재하는 아미노산 측쇄를 보다 작은 측쇄(hole;공극)로 치환함으로써, 돌기가 공극 내에 배치되도록 하여 이종 H쇄 형성의 촉진 및 동종 H쇄 형성의 저해를 일으키는 방법이다. 또한 각각의 IgG H쇄의 CH3영역에 다른 전하를 도입하는 방법도 보고되어 있다(특허문헌 2). 구체적으로는, 한쪽 H쇄의 CH3영역에 존재하는 아미노산을 양전하를 갖는 아미노산으로 치환하고, 다른 한쪽 H쇄의 CH3영역에 존재하는 아미노산을 음전하를 갖는 아미노산으로 치환함으로써, 이종 H쇄 형성의 촉진 및 동종 H쇄 형성의 저해를 일으키는 방법이다. 한편 H쇄와 L쇄의 조합을 제어하는 기술도 보고되어 있다(비특허문헌 6). 본 수법은 항체의 한쪽 Fab로서 L쇄 불변영역(CL)과 H쇄 CH1영역을 바꿔 넣은 것을 사용함으로써 목적으로 하는 H쇄와 L쇄의 조합을 효율적으로 야기하는 수법이다. 또한 이 밖에는 양쪽 Fab에서 공통된 L쇄를 사용하는 수법도 존재한다. 이는 공통의 L쇄를 사용함으로써 세포에 도입하는 L쇄 유전자를 1종류로 하여, H쇄와 L쇄의 조합을 고려할 필요 없이 이중특이성 항체를 얻을 수 있는 것이다. 현재는 이종 H쇄 형성 기술, H쇄 L쇄 조합 제어기술을 조합함으로써 높은 효율로 이중특이성 항체를 형성할 수 있다. 그러나, H쇄와 L쇄의 조합을 완전히 제어하는 것은 어려워 복잡한 분자 설계가 된다. 또한 공통의 L쇄에서 2종류의 항원에 대해 높은 친화성을 유지하는 것은 난이도가 높다는 문제도 있다. Although the production of an IgG-type bispecific antibody is difficult due to the low efficiency at which the target bispecific antibody is obtained and the difficulty in purification, several knowledge regarding efficient production have been reported (Non-Patent Document 3). For example, when a total of four genes, including H chain and L chain genes constituting IgG having two types of variable regions, are introduced into cells and secreted by coexpression, the covalent bond between the two types of H chains or H chain Since the non-covalent bond between the L chain and the L chain occurs randomly, the ratio of the target bispecific antibody is very small, and the production efficiency is significantly reduced. As a means of solving this problem, it has been reported that by performing amino acid substitution in the CH3 region of the IgG H chain, it is possible to preferentially secrete a heterologous combination of IgG to the H chain (Patent Document 1 and Non-Patent Documents 4 and 5). In this method, the amino acid side chain present in the CH3 region of one H chain is substituted with a larger side chain (knob), and the amino acid side chain present in the CH3 region of the other H chain is substituted with a smaller side chain (hole). This is a method of causing the protrusions to be arranged in the voids to promote the formation of heterogeneous H chains and inhibit the formation of homologous H chains. Also, a method of introducing different charges into the CH3 region of each IgG H chain has been reported (Patent Document 2). Specifically, by substituting an amino acid present in the CH3 region of one H chain with an amino acid having a positive charge, and substituting an amino acid present in the CH3 region of the other H chain with an amino acid having a negative charge, promoting and It is a method of causing inhibition of homologous H chain formation. On the other hand, a technique for controlling the combination of H chain and L chain has also been reported (Non-Patent Document 6). This technique is a technique for efficiently generating the desired combination of H chain and L chain by using one of the antibody Fab in which the L chain constant region (CL) and the H chain CH1 region are exchanged. In addition, there is also a method of using a common L chain in both Fabs. By using a common L chain, a bispecific antibody can be obtained without considering the combination of the H chain and the L chain by making one type of L chain gene to be introduced into the cell. Currently, a bispecific antibody can be formed with high efficiency by combining a heterogeneous H chain formation technology and a H chain L chain combination control technology. However, it is difficult to completely control the combination of H and L chains, resulting in complex molecular design. In addition, there is also a problem that it is difficult to maintain high affinity for two types of antigens in a common L chain.

한편 상기와 같은 유전자 재조합법이 아니라 사전에 별도로 조제한 단일클론 항체끼리를 사용하여 이중특이성 항체를 제작하는 방법으로서 Fab Arm Exchange로 불리는 수법이 보고되어 있다. 이는 생체 내에서 IgG4의 반분자가 내인성 IgG4의 반분자와 교환하여 이중특이성 항체(Bispecific 항체；BiAb)를 생성한다는 지견(비특허문헌 7)에 기초하여 개발된 기술로, in vitro에서 2종류의 천연형 인간 IgG4를 혼합함으로써 이중특이성 항체가 생산되는 것(특허문헌 3), 더 나아가서는 환원 조건하에 있어서 보다 효율적으로 반응이 일어나는 것이 보고되어 있다(비특허문헌 8). 또한 IgG4에 특이적인 힌지영역의 228번째와 CH3영역의 409번째 2개소가 이 반응에 중요한 아미노산 잔기인 것이 특정되어, IgG1이라도 이 2개소를 IgG4형의 아미노산으로 치환함으로써 IgG4와 동등한 효율로 반응이 일어나는 것이 발견되어 있다(특허문헌 4). Fab Arm Exchange는 일반적인 방법으로 제작된 단일클론 항체끼리를 in vitro에서 혼합하는 것만으로 목적으로 하는 이중특이성 항체가 얻어지기 때문에 범용성이 높다. 그러나 반분자 교환 반응은 랜덤으로 일어나기 때문에, 2종류의 항체를 혼합하여 얻어지는 이중특이성 항체는 이론상 시스템 내에 존재하는 전 항체량의 50%가 된다. 이 때문에 이중특이성 항체 생성률을 개선하는 방법이 검토되어, 2종류의 항체에 비대칭인 아미노산 개변, 즉, 한쪽 H쇄에 K409R의 개변 및 다른 한쪽 H쇄에 F405L의 개변을 도입함으로써 반응률이 크게 향상되는 것이 보고되어 있는데 반응 효율은 95% 정도에 그치고 있다(특허문헌 5, 비특허문헌 9). 이중특이성 항체의 효율적이고 안정한 제조에 있어서는 정제의 간편함과 로트 간의 불균일성을 가능한 한 저감시키는 것이 필요 불가결하기 때문에, 보다 높은 반응 효율을 실현하는 우수한 수법의 개발이 요망된다. On the other hand, a method called Fab Arm Exchange has been reported as a method for producing a bispecific antibody using monoclonal antibodies prepared separately in advance rather than the above-described genetic recombination method. This is a technology developed based on the knowledge (Non-Patent Document 7) that a half molecule of IgG4 is exchanged with an half molecule of endogenous IgG4 in vivo to generate a bispecific antibody (BiAb). It has been reported that a bispecific antibody is produced by mixing native human IgG4 (Patent Document 3), and that the reaction occurs more efficiently under reducing conditions (Non-Patent Document 8). In addition, it was determined that the two sites 228 and 409 of the hinge region specific to IgG4 are important amino acid residues for this reaction, and by substituting these two sites with an IgG4 amino acid even for IgG1, the reaction can be performed with the same efficiency as that of IgG4. It has been found to occur (Patent Document 4). Fab arm exchange is highly versatile because a target bispecific antibody can be obtained only by in vitro mixing of monoclonal antibodies produced by a general method. However, since the half-molecular exchange reaction occurs randomly, the bispecific antibody obtained by mixing two types of antibodies is theoretically 50% of the total amount of antibodies present in the system. For this reason, a method for improving the bispecific antibody production rate has been studied, and the reaction rate is greatly improved by introducing asymmetric amino acid modifications to two types of antibodies, that is, K409R modification in one H chain and F405L modification in the other H chain. It has been reported that the reaction efficiency is only about 95% (Patent Document 5, Non-Patent Document 9). In the efficient and stable production of bispecific antibodies, it is essential to simplify purification and to reduce the non-uniformity between lots as much as possible.

WO1996/027011WO1996/027011 WO2006/106905WO2006/106905 WO2005/062916WO2005/062916 WO2008/119353WO2008/119353 WO2011/131746WO2011/131746

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본 발명은 이러한 상황을 감안하여 이루어진 것으로, 그 목적은 이종 다량체의 효율적이고 또한 안정한 제조를 위해 환원 상황하에 있어서 폴리펩티드의 헤테로화를 촉진하여 목적하는 이종 다량체를 얻는, 보다 반응 효율이 높은 우수한 수법을 제공하는 것이다. The present invention has been made in view of this situation, and its object is to promote heterogeneity of a polypeptide under reducing conditions for efficient and stable production of a heteromultimer to obtain a desired heteromultimer, and to obtain an excellent reaction efficiency with higher reaction efficiency. to provide a method.

본 발명자들은 이종 다량체에 제공하는 폴리펩티드로서 Fc영역을 갖는 폴리펩티드를 선택하여, Fc영역의 해리 및 회합을 제어하는 방법에 대해서 예의 연구를 행하였다. 그 결과, 중쇄 CH3영역에 존재하는 특정 아미노산을 치환함으로써 환원 상황하에 있어서 Fc영역의 해리의 촉진 및 회합의 제어가 가능하여, 종래 기술과 비교하여 효율적으로 목적하는 헤테로 분자가 형성되는 것을 발견하였다. The present inventors selected a polypeptide having an Fc region as a polypeptide to be provided to a heteromultimer, and conducted intensive studies on a method for controlling dissociation and association of the Fc region. As a result, it was found that by substituting a specific amino acid present in the heavy chain CH3 region, it was possible to promote the dissociation and control the association of the Fc region under reducing conditions, thereby forming the desired hetero molecule more efficiently compared to the prior art.

본 발명은 이러한 지견(知見)에 기초하는 것으로, 구체적으로는 아래의 [1] 내지 [25]를 제공한다. The present invention is based on such knowledge, and specifically, the following [1] to [25] are provided.

[1] a) 제1 항원 결합 활성을 갖고, Fc영역을 포함하는 제1 폴리펩티드의 호모체를 제공하는 단계, [1] a) having a first antigen-binding activity and providing a homo body of a first polypeptide comprising an Fc region;

b) 상기 제1 항원 결합 활성과 다른 제2 항원 결합 활성을 갖고, Fc영역을 포함하는 제2 폴리펩티드의 호모체를 제공하는 단계, b) having a second antigen-binding activity different from the first antigen-binding activity, providing a homo body of a second polypeptide comprising an Fc region;

c) 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키는 것을 가능하게 하는 환원 조건하에서, 상기 제1 폴리펩티드의 호모체 및 상기 제2 폴리펩티드의 호모체를 함께 인큐베이트하는 단계, 및 c) incubating the homoform of the first polypeptide and the homoform of the second polypeptide together under reducing conditions that allow the cysteine in the hinge region to cause isomerization of the disulfide bond, and

d) 제1 및 제2 폴리펩티드를 포함하는 이종 다량체를 얻는 단계d) obtaining a heteromultimer comprising the first and second polypeptides;

를 포함하고, including,

상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서, In the CH3 region included in the Fc region of the first and/or second polypeptide,

(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기(1) amino acid residues at positions 356 and 439 by EU numbering

(2) EU 넘버링에 의한 357번 위치 및 370번 위치의 아미노산 잔기(2) amino acid residues at positions 357 and 370 by EU numbering

(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기(3) amino acid residues at positions 399 and 409 by EU numbering

에 나타내는 아미노산 잔기의 조(set)로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동종의 전하를 가지며, 1 to 3 sets of amino acid residues selected from the set of amino acid residues shown in

상기 제1 폴리펩티드의 CH3영역 및 상기 제2 폴리펩티드의 CH3영역 양쪽에 있어서, 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 같은 조의 아미노산 잔기가 각각 동종의 전하를 갖는 경우, 상기 제2 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기가 상기 제1 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기와는 반대의 전하를 갖는, In both the CH3 region of the first polypeptide and the CH3 region of the second polypeptide, when the amino acid residues of the same group among the groups of amino acid residues shown in (1) to (3) have the same charge, the second The pair of amino acid residues in the CH3 region of the polypeptide has a charge opposite to that of the pair of amino acid residues in the CH3 region of the first polypeptide;

이종 다량체를 제조하기 위한 방법. A method for preparing heteromultimers.

[2] [1]의 단계 a)에 있어서 제1 폴리펩티드와 다량체를 형성하는 제3 폴리펩티드를 제공하는 단계를 포함하고, 또한 단계 b)에 있어서 제2 폴리펩티드와 다량체를 형성하는 제4 폴리펩티드를 제공하는 단계를 포함하는, [1]에 기재된 제조방법. [2] a fourth polypeptide comprising the step of providing a third polypeptide that multimers with the first polypeptide in step a) of [1], and that forms a multimer with the second polypeptide in step b) The manufacturing method according to [1], comprising the step of providing.

[3] 상기 동종의 전하를 갖는 아미노산 잔기가 아래의 (A) 또는 (B) 중 어느 하나의 군에 포함되는 하나 이상의 아미노산 잔기로부터 선택되는, [1] 또는 [2]에 기재된 제조방법：[3] The production method according to [1] or [2], wherein the amino acid residue having the same charge is selected from one or more amino acid residues included in any one of the following groups (A) or (B):

(A) 글루타민산(E), 아스파라긴산(D)； (A) glutamic acid (E), aspartic acid (D);

(B) 리신(K), 아르기닌(R), 히스티딘(H). (B) Lysine (K), arginine (R), histidine (H).

[4] 제1 및 제2 폴리펩티드에 있어서 각각 동종의 전하를 갖는 아미노산 잔기의 조가 아래의 (1)~(4) 중 어느 한 조의 아미노산 잔기의 조인, [1] 내지 [3] 중 어느 한 항에 기재된 제조방법：[4] Any one of [1] to [3], wherein in the first and second polypeptides, the set of amino acid residues each having the same charge is a combination of any one of the following sets of amino acid residues (1) to (4) The manufacturing method described in:

(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기(1) amino acid residues at positions 356 and 439 by EU numbering

(2) EU 넘버링에 의한 357번 위치 및 370번 위치의 아미노산 잔기(2) amino acid residues at positions 357 and 370 by EU numbering

(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기(3) amino acid residues at positions 399 and 409 by EU numbering

(4) (i) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기, 및(4) (i) the amino acid residues at positions 399 and 409 by EU numbering, and

(ii) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기. (ii) amino acid residues at positions 356 and 439 by EU numbering.

[5] 제1 및 제2 폴리펩티드에 있어서 각각 동종의 전하를 갖는 아미노산 잔기의 조가 아래의 조의 아미노산 잔기의 조인, [1] 내지 [4] 중 어느 한 항에 기재된 제조방법：[5] The production method according to any one of [1] to [4], wherein in the first and second polypeptides, the group of amino acid residues having the same charge, respectively, is a group of amino acid residues of the following group:

(i) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기, 및(i) the amino acid residues at positions 399 and 409 by EU numbering, and

(ii) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기. (ii) amino acid residues at positions 356 and 439 by EU numbering.

[6] 제1 및/또는 제2 폴리펩티드에 있어서 제1 및/또는 제2 폴리펩티드의 CH3영역의 안정성이 불안정해지도록 아미노산이 개변되어 있는, [1] 내지 [5] 중 어느 한 항에 기재된 제조방법. [6] The preparation according to any one of [1] to [5], wherein, in the first and/or second polypeptide, the amino acid is modified so that the stability of the CH3 region of the first and/or second polypeptide is unstable. Way.

[7] 제1 및/또는 제2 폴리펩티드에 있어서 EU 넘버링에 의한 397번 위치 및/또는 392번 위치의 아미노산이 개변되어 있는, [1] 내지 [6] 중 어느 한 항에 기재된 제조방법. [7] The production method according to any one of [1] to [6], wherein, in the first and/or second polypeptide, the amino acid at position 397 and/or position 392 according to EU numbering is modified.

[8] 제1 및/또는 제2 폴리펩티드의 Fc영역이 IgG1, IgG2, IgG3 또는 IgG4 타입인, [1] 내지 [7] 중 어느 한 항에 기재된 제조방법. [8] The production method according to any one of [1] to [7], wherein the Fc region of the first and/or second polypeptide is an IgG1, IgG2, IgG3 or IgG4 type.

[9] 제1 및/또는 제2 폴리펩티드의 Fc영역이 마우스 유래의 Fc영역인, [1] 내지 [7] 중 어느 한 항에 기재된 제조방법. [9] The production method according to any one of [1] to [7], wherein the Fc region of the first and/or second polypeptide is a mouse-derived Fc region.

[10] 상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서, [10] In the CH3 region included in the Fc region of the first and/or second polypeptide,

(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기(1) amino acid residues at positions 356 and 439 by EU numbering

(2) EU 넘버링에 의한 360번 위치 및 371번 위치의 아미노산 잔기(2) amino acid residues at positions 360 and 371 by EU numbering

(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기(3) amino acid residues at positions 399 and 409 by EU numbering

에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동종의 전하를 갖고, 1 to 3 sets of amino acid residues selected from the group of amino acid residues shown in

상기 제1 폴리펩티드의 CH3영역 및 상기 제2 폴리펩티드의 CH3영역 양쪽에 있어서, 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 같은 조의 아미노산 잔기가 각각 동종의 전하를 갖는 경우, 상기 제2 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기가 상기 제1 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기와는 반대의 전하를 갖는, [9]에 기재된 이종 다량체를 제조하기 위한 방법.In both the CH3 region of the first polypeptide and the CH3 region of the second polypeptide, when the amino acid residues of the same group among the groups of amino acid residues shown in (1) to (3) have the same charge, the second The method for producing a heteromultimer according to [9], wherein the set of amino acid residues in the CH3 region of the polypeptide has a charge opposite to that of the set of amino acid residues in the CH3 region of the first polypeptide.

[11] a) 제1 항원 결합 활성을 갖고, Fc영역을 포함하는 제1 폴리펩티드의 호모체를 제공하는 단계, [11] a) having a first antigen-binding activity and providing a homo body of a first polypeptide comprising an Fc region;

b) 상기 제1 항원 결합 활성과 다른 제2 항원 결합 활성을 갖고, Fc영역을 포함하는 제2 폴리펩티드의 호모체를 제공하는 단계, b) having a second antigen-binding activity different from the first antigen-binding activity, providing a homo body of a second polypeptide comprising an Fc region;

c) 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키는 것을 가능하게 하는 환원 조건하에서, 상기 제1 폴리펩티드의 호모체 및 상기 제2 폴리펩티드의 호모체를 함께 인큐베이트하는 단계, 및 c) incubating the homoform of the first polypeptide and the homoform of the second polypeptide together under reducing conditions that allow the cysteine in the hinge region to cause isomerization of the disulfide bond, and

d) 제1 및 제2 폴리펩티드를 포함하는 이종 다량체를 얻는 단계d) obtaining a heteromultimer comprising the first and second polypeptides;

를 포함하고, including,

상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서의 EU 넘버링에 의한 397번 위치 및/또는 392번 위치의 아미노산이 개변되어 있는, The amino acid at position 397 and/or position 392 by EU numbering in the CH3 region contained in the Fc region of the first and/or second polypeptide is altered,

이종 다량체를 제조하기 위한 방법. A method for preparing heteromultimers.

[12] 제1 및/또는 제2 폴리펩티드에 있어서,[12] The first and/or second polypeptide,

EU 넘버링에 의한 397번 위치가 Met(M), Phe(F) 또는 Tyr(Y)로 개변되어 있고, 및/또는 Position 397 by EU numbering is modified with Met (M), Phe (F) or Tyr (Y), and / or

EU 넘버링에 의한 392번 위치가 Asp(D), Glu(E), Thr(T), Val(V) 또는 Ile(I)로 개변되어 있는Position 392 by EU numbering is modified with Asp (D), Glu (E), Thr (T), Val (V) or Ile (I)

[1] 내지 [11] 중 어느 한 항에 기재된 제조방법. The production method according to any one of [1] to [11].

[13] 제1 및/또는 제2 폴리펩티드에 있어서 EU 넘버링에 의한 397번 위치가 Phe(F) 또는 Tyr(Y)로 개변되어 있는, [1] 내지 [12] 중 어느 한 항에 기재된 제조방법. [13] The production method according to any one of [1] to [12], wherein in the first and/or second polypeptide, position 397 by EU numbering is modified with Phe (F) or Tyr (Y). .

[14] 제1 폴리펩티드에 있어서 EU 넘버링에 의한 356번 위치가 Lys(K)로 개변되어 있고, 또한 EU 넘버링에 의한 397번 위치가 Phe(F) 또는 Tyr(Y)로 개변되어 있으며, 제2 폴리펩티드에 있어서 EU 넘버링에 의한 397번 위치가 Phe(F) 또는 Tyr(Y)로 개변되어 있고, 또한 EU 넘버링에 의한 439번 위치가 Glu(E)로 개변되어 있는, [1] 내지 [13] 중 어느 한 항에 기재된 제조방법. [14] In the first polypeptide, position 356 by EU numbering is modified to Lys (K), and position 397 by EU numbering is modified to Phe (F) or Tyr (Y), and the second In the polypeptide, position 397 by EU numbering is modified with Phe (F) or Tyr (Y), and position 439 by EU numbering is modified with Glu (E), [1] to [13] The manufacturing method in any one of Claims.

[15] 상기 단계 a) 및 b)가 제1 폴리펩티드의 호모체를 생산하는 세포주와 제2 폴리펩티드의 호모체를 생산하는 세포주를 혼합함으로써 실시되고, 상기 단계 c)가 당해 배양상청 중에서 실시되는, [1] 내지 [14] 중 어느 한 항에 기재된 제조방법. [15] The steps a) and b) are carried out by mixing a cell line producing a homo body of the first polypeptide and a cell line producing a homo body of the second polypeptide, and the step c) is carried out in the culture supernatant; The production method according to any one of [1] to [14].

[16] 상기 이종 다량체가 다중특이성 항체 또는 헤테로 Fc 융합 단백질인, [1] 내지 [15] 중 어느 한 항에 기재된 제조방법. [16] The production method according to any one of [1] to [15], wherein the heteromultimer is a multispecific antibody or a hetero Fc fusion protein.

[17] 상기 이종 다량체가 이중특이성 항체인, [1] 내지 [16] 중 어느 한 항에 기재된 제조방법. [17] The production method according to any one of [1] to [16], wherein the heteromultimer is a bispecific antibody.

[18] [1] 또는 [11]에 기재된 단계 c)가 환원제와의 접촉을 포함하는, [1] 내지 [17] 중 어느 한 항에 기재된 제조방법. [18] The production method according to any one of [1] to [17], wherein step c) according to [1] or [11] includes contacting with a reducing agent.

[19] 단계 c)가 글루타티온, L-시스테인, 디티오트레이톨, β-메르캅토-에탄올, TCEP 및 2-MEA로 이루어진 군으로부터 선택되는 작용물질의 첨가를 포함하는, [18]에 기재된 제조방법. [19] The preparation according to [18], wherein step c) comprises the addition of an agent selected from the group consisting of glutathione, L-cysteine, dithiothreitol, β-mercapto-ethanol, TCEP and 2-MEA Way.

[20] 단계 c)가 글루타티온, 2-MEA로부터 선택되는 작용물질의 첨가를 포함하는, [19]에 기재된 제조방법. [20] The production method according to [19], wherein step c) comprises adding an agonist selected from glutathione and 2-MEA.

[21] [1] 내지 [20] 중 어느 한 항에 기재된 방법으로 제조된 이종 다량체. [21] A heteromultimer produced by the method according to any one of [1] to [20].

[22] 이중특이성 항체인, [21]에 기재된 이종 다량체. [22] The heteromultimer according to [21], which is a bispecific antibody.

[23] [21] 또는 [22]에 기재된 이종 다량체 및 의약적으로 허용되는 담체를 포함하는 조성물. [23] A composition comprising the heteromultimer according to [21] or [22] and a pharmaceutically acceptable carrier.

[24] 제1 항원 결합 활성을 갖고 또한 제1 Fc영역을 포함하는 제1 폴리펩티드와, 그 제1 항원 결합 활성과는 다른 제2 항원 결합 활성을 갖고 또한 제2 Fc영역을 포함하는 제2 폴리펩티드를 포함하는 이종 다량체로서, [24] a first polypeptide having a first antigen-binding activity and comprising a first Fc region, and a second polypeptide having a second antigen-binding activity different from the first antigen-binding activity and comprising a second Fc region As a heteromultimer comprising a,

상기 제1 폴리펩티드의 호모체 및 상기 제2 폴리펩티드의 호모체를 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키는 것을 가능하게 하는 환원 조건하에서 함께 인큐베이트함으로써 상기 이종 다량체가 얻어지고, The heteromultimer is obtained by incubating the homoform of the first polypeptide and the homoform of the second polypeptide together under reducing conditions enabling cysteine in the hinge region to cause isomerization of the disulfide bond,

상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서, In the CH3 region included in the Fc region of the first and/or second polypeptide,

(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기(1) amino acid residues at positions 356 and 439 by EU numbering

(2) EU 넘버링에 의한 357번 위치 및 370번 위치의 아미노산 잔기(2) amino acid residues at positions 357 and 370 by EU numbering

(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기(3) amino acid residues at positions 399 and 409 by EU numbering

에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동정의 전하를 가지며, 1 to 3 sets of amino acid residues selected from the group of amino acid residues shown in

상기 제1 폴리펩티드의 CH3영역 및 상기 제2 폴리펩티드의 CH3영역 양쪽에 있어서, 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 같은 조의 아미노산 잔기가 각각 동종의 전하를 갖는 경우, 상기 제2 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기가 상기 제1 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기와는 반대의 전하를 갖고, In both the CH3 region of the first polypeptide and the CH3 region of the second polypeptide, when the amino acid residues of the same group among the groups of amino acid residues shown in (1) to (3) have the same charge, the second The pair of amino acid residues in the CH3 region of the polypeptide has a charge opposite to that of the pair of amino acid residues in the CH3 region of the first polypeptide;

상기 제1 및/또는 제2 폴리펩티드에 있어서 상기 제1 및/또는 제2 폴리펩티드의 CH3영역의 안정성이 불안정해지도록 아미노산이 개변되어 있는, In the first and / or second polypeptide, the amino acid is modified so that the stability of the CH3 region of the first and / or second polypeptide is unstable,

상기 이종 다량체. The heteromultimer.

[25] a) 제1 항원 결합 활성을 갖고, Fc영역을 포함하는 제1 폴리펩티드의 호모체를 제공하는 단계, [25] a) having a first antigen-binding activity and providing a homo body of a first polypeptide comprising an Fc region;

b) 상기 제1 항원 결합 활성과 다른 제2 항원 결합 활성을 갖고, Fc영역을 포함하는 제2 폴리펩티드의 호모체를 제공하는 단계, b) having a second antigen-binding activity different from the first antigen-binding activity, providing a homo body of a second polypeptide comprising an Fc region;

c) 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키는 것을 가능하게 하는 환원 조건하에서, 상기 제1 폴리펩티드의 호모체 및 상기 제2 폴리펩티드의 호모체를 함께 인큐베이트하는 단계, 및 c) incubating the homoform of the first polypeptide and the homoform of the second polypeptide together under reducing conditions that allow the cysteine in the hinge region to cause isomerization of the disulfide bond, and

d) 제1 및 제2 폴리펩티드를 포함하는 이종 다량체를 얻는 단계d) obtaining a heteromultimer comprising the first and second polypeptides;

를 포함하는 방법으로 제조된 이종 다량체로서, 상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서, As a heteromultimer prepared by a method comprising a, in the CH3 region included in the Fc region of the first and / or second polypeptide,

(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기(1) amino acid residues at positions 356 and 439 by EU numbering

(2) EU 넘버링에 의한 357번 위치 및 370번 위치의 아미노산 잔기(2) amino acid residues at positions 357 and 370 by EU numbering

(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기(3) amino acid residues at positions 399 and 409 by EU numbering

에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동정의 전하를 갖고, 1 to 3 sets of amino acid residues selected from the group of amino acid residues shown in

상기 제1 폴리펩티드의 CH3영역 및 상기 제2 폴리펩티드의 CH3영역 양쪽에 있어서, 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 같은 조의 아미노산 잔기가 각각 동종의 전하를 갖는 경우, 상기 제2 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기가 상기 제1 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기와는 반대의 전하를 가지며,In both the CH3 region of the first polypeptide and the CH3 region of the second polypeptide, when the amino acid residues of the same group among the groups of amino acid residues shown in (1) to (3) have the same charge, the second The pair of amino acid residues in the CH3 region of the polypeptide has a charge opposite to that of the pair of amino acid residues in the CH3 region of the first polypeptide;

상기 제1 및/또는 제2 폴리펩티드에 있어서 상기 제1 및/또는 제2 폴리펩티드의 CH3영역의 안정성이 불안정해지도록 아미노산이 개변되어 있는, In the first and / or second polypeptide, the amino acid is modified so that the stability of the CH3 region of the first and / or second polypeptide is unstable,

상기 이종 다량체. The heteromultimer.

본 발명에서는 중쇄 CH3영역에 존재하는 특정 아미노산을 치환함으로써, 환원 상황하에 있어서 Fc영역의 해리의 촉진 및 회합의 제어가 가능하여, 종래 기술과 비교하여 효율적으로 목적하는 헤테로 분자가 형성되는 제조방법이 제공되었다. In the present invention, by substituting a specific amino acid present in the heavy chain CH3 region, it is possible to promote the dissociation and control the association of the Fc region under reducing conditions, and a method for efficiently forming the desired hetero molecule compared to the prior art. was provided

본 발명의 방법을 이용함으로써, 종래 기술과 비교하여 이중특이성 항체의 정제 시 간편함의 향상과 로트 간의 불균일성을 가능한 한 저감시키는 것이 가능해졌다. By using the method of the present invention, compared with the prior art, it became possible to improve the convenience in purifying the bispecific antibody and to reduce the non-uniformity between lots as much as possible.

본 발명의 이종 다량체의 제조방법은 중쇄 CH3영역의 아미노산 잔기를 개변하는 것을 특징으로 한다. 이들 영역에 본 발명의 아미노산 잔기의 개변을 도입함으로써 폴리펩티드 간의 해리 및 회합이 촉진되어, 종래 기술과 비교하여 효율적으로 목적하는 이종 다량체를 얻을 수 있다.The method for producing a heteromultimer of the present invention is characterized in that the amino acid residues of the heavy chain CH3 region are modified. By introducing modifications of the amino acid residues of the present invention into these regions, dissociation and association between polypeptides is promoted, and a desired heteromultimer can be obtained more efficiently than in the prior art.

도 1은 이온 교환 크로마토그래피에 의한 Fab Arm Exchange 반응산물의 해석결과를 나타내는 도면이다. 도면 중 「BiAb」는 정제한 Bispecific 항체(이중특이성 항체)를, 「H54 homo」는 H54/L28의 가변영역을 갖는 단일클론 항체를, 「MRA homo」는 MRAH/MRAL의 가변영역을 갖는 단일클론 항체를 나타낸다. 도면 중에 백분율로 나타낸 수치는 이중특이성 항체 생성률을 나타내고, 이중특이성 항체에 대응하는 피크의 면적을 시스템 중에 존재하는 전 항체의 면적으로 나누고, 100을 곱하여 산출한 것이다.
도 2는 이온 교환 크로마토그래피에 의한 Fab Arm Exchange 반응산물의 해석결과를 나타내는 도면이다. MRAH-G1drP1/MRAL-k0와 H54-G1drN1/L28-k0를 호모체로서 사용하고, 3종류의 환원 조건하에서 반응시킨 결과를 나타내는 도면이다. 도면 중에 백분율로 나타낸 수치는 이중특이성 항체 생성률을 나타내고, 이중특이성 항체에 대응하는 피크의 면적을 시스템 중에 존재하는 전 항체의 면적으로 나누고, 100을 곱하여 산출한 것이다.
도 3은 5 mM GSH를 환원제로서 사용했을 때의 Fab Arm Exchange에 있어서의 이중특이성 항체 생성률과, 사용한 호모체의 CH3 안정성의 상관관계를 나타내는 도면이다. 도면 중 「2종류의 호모체 중 CH3의 Tm이 높은 쪽의 값」이란, 반응에 사용하는 2개의 호모체 중 CH3의 Tm이 높은, 즉 CH3가 보다 안정한 호모체의 CH3의 Tm을 의미한다.
도 4는 인간 IgG1의 V397 주변의 입체구조(PDB code 3DO3)를 나타내는 도면이다.
도 5는 25 mM 2MEA를 환원제로서 사용했을 때의 Fab Arm Exchange에 있어서의 이중특이성 항체 생성률과, 사용한 호모체의 CH3 안정성의 상관관계를 나타내는 도면이다. 도면 중 「2종류의 호모체 중 CH3의 Tm이 높은 쪽의 값」이란, 반응에 사용하는 2개의 호모체 중 CH3의 Tm이 높은, 즉 CH3가 보다 안정한 호모체의 CH3의 Tm을 의미한다.
도 6은 이온 교환 크로마토그래피에 의한 Fab Arm Exchange 반응산물의 해석결과를 나타내는 도면이다. MRAH-G1dP17/MRAL-k0와 H54-G1dN17/L28-k0를 호모체로서 사용하여, 다른 반응 시간으로 반응시킨 결과를 나타내는 도면이다. 도면 중에 백분율로 나타낸 수치는 이중특이성 항체 생성률을 나타내고, 이중특이성 항체에 대응하는 피크의 면적을 시스템 중에 존재하는 전 항체의 면적으로 나누고, 100을 곱하여 산출한 것이다.
도 7은 이온 교환 크로마토그래피에 의한 Fab Arm Exchange 반응산물의 해석결과를 나타내는 도면이다. MRAH-G1mrP1/MRAL-k0와 H54-G1mrN1/L28-k0를 호모체로서 사용하여, 세포 배양상청 중에서 반응시킨 결과를 나타내는 도면이다. 도면 중에 백분율로 나타낸 수치는 이중특이성 항체 생성률을 나타내고, 이중특이성 항체에 대응하는 피크의 면적을 시스템 중에 존재하는 전 항체의 면적으로 나누고, 100을 곱하여 산출한 것이다.
도 8은 마우스 IgG1의 CH3 도메인의 상호작용 계면 주변의 입체구조를 나타내는 도면이다(PDB code 1IGY).
도 9는 CE-IEF에 의한 마우스 IgG형 Fab Arm Exchange 반응산물의 해석결과를 나타내는 도면이다. 도면 중에 백분율로 나타낸 수치는 이중특이성 항체 생성률을 나타내고, 이중특이성 항체에 대응하는 피크의 면적을 시스템 중에 존재하는 전 항체의 면적으로 나누고, 100을 곱하여 산출한 것이다.
도 10은 항인간 글리피칸 3/항인간 CD3 이중특이성 항체의 세포상해활성을 비교한 도면이다. 도 10-1은 인간 IgG형 Fab Arm Exchange를 사용하여, 도 10-2는 마우스 IgG형 Fab Arm Exchange를 사용하여 제작한 이중특이성 항체와, CrossMab 기술에 의해 제작한 이중특이성 항체를 비교한 것이다.
도 11은 인간 IgG형 Fab Arm Exchange에 의해 제작한 항인간 글리피칸 3/항인간 CD3 이중특이성 항체 및 Knobs-into-Holes 기술에 의해 제작한 이중특이성 항체의 정상 마우스에 있어서의 혈중 농도 추이를 나타낸 도면이다.
도 12는 마우스 IgG형 Fab Arm Exchange에 의해 제작한 항인간 IL-6 수용체 항체와, 천연형 마우스 IgG1의 서열을 갖는 항인간 IL-6 수용체 항체의 혈중 농도 추이를 나타낸 도면이다. 1 is a view showing the analysis result of the Fab Arm Exchange reaction product by ion exchange chromatography. In the figure, “BiAb” denotes a purified bispecific antibody (bispecific antibody), “H54 homo” denotes a monoclonal antibody having a variable region of H54/L28, and “MRA homo” denotes a monoclonal antibody having a variable region of MRAH/MRAL. indicates an antibody. Numerical values expressed as percentages in the figure represent the bispecific antibody production rate, and are calculated by dividing the area of the peak corresponding to the bispecific antibody by the area of all antibodies present in the system, and multiplying by 100.
2 is a view showing the analysis result of the Fab Arm Exchange reaction product by ion exchange chromatography. It is a figure which shows the result of reacting under three types of reducing conditions using MRAH-G1drP1/MRAL-k0 and H54-G1drN1/L28-k0 as homo bodies. Numerical values expressed as percentages in the figure represent the bispecific antibody production rate, and are calculated by dividing the area of the peak corresponding to the bispecific antibody by the area of all antibodies present in the system, and multiplying by 100.
Fig. 3 is a diagram showing the correlation between the bispecific antibody production rate in Fab arm exchange when 5 mM GSH is used as a reducing agent, and the CH3 stability of the homoform used. In the figure, "the value of the higher Tm of CH3 among the two types of homoforms" means the Tm of CH3 of the homo form having the higher Tm of CH3, that is, the more stable CH3 among the two homoforms used for the reaction.
4 is a diagram showing the three-dimensional structure (PDB code 3DO3) around V397 of human IgG1.
Fig. 5 is a diagram showing the correlation between the bispecific antibody production rate in Fab arm exchange when 25 mM 2MEA is used as a reducing agent, and the CH3 stability of the homoform used. In the figure, "the value of the higher Tm of CH3 among the two types of homoforms" means the Tm of CH3 of the homo form having the higher Tm of CH3, that is, the more stable CH3 among the two homoforms used for the reaction.
6 is a view showing the analysis result of the Fab Arm Exchange reaction product by ion exchange chromatography. It is a figure showing the result of reaction with different reaction times using MRAH-G1dP17/MRAL-k0 and H54-G1dN17/L28-k0 as homo bodies. Numerical values expressed as percentages in the figure represent the bispecific antibody production rate, and are calculated by dividing the area of the peak corresponding to the bispecific antibody by the area of all antibodies present in the system, and multiplying by 100.
7 is a view showing the analysis result of the Fab Arm Exchange reaction product by ion exchange chromatography. It is a figure showing the result of reaction in cell culture supernatant using MRAH-G1mrP1/MRAL-k0 and H54-G1mrN1/L28-k0 as homo bodies. Numerical values expressed as percentages in the figure represent the bispecific antibody production rate, and are calculated by dividing the area of the peak corresponding to the bispecific antibody by the area of all antibodies present in the system, and multiplying by 100.
8 is a diagram showing the three-dimensional structure around the interaction interface of the CH3 domain of mouse IgG1 (PDB code 1IGY).
9 is a diagram showing the analysis results of the mouse IgG-type Fab Arm Exchange reaction product by CE-IEF. Numerical values expressed as percentages in the figure represent the bispecific antibody production rate, and are calculated by dividing the area of the peak corresponding to the bispecific antibody by the area of all antibodies present in the system, and multiplying by 100.
10 is a diagram comparing the cytotoxic activity of anti-human glypican 3/anti-human CD3 bispecific antibodies. FIG. 10-1 is a comparison of a bispecific antibody produced using a human IgG-type Fab Arm Exchange, and FIG. 10-2 is a bispecific antibody produced using a mouse IgG-type Fab Arm Exchange, and a bispecific antibody produced by CrossMab technology.
Fig. 11 is an anti-human glypican 3/anti-human CD3 bispecific antibody produced by human IgG-type Fab Arm Exchange and a bispecific antibody produced by Knobs-into-Holes technology in normal mice. It is a drawing.
Fig. 12 is a diagram showing the transition of blood concentration of an anti-human IL-6 receptor antibody produced by mouse IgG-type Fab Arm Exchange and an anti-human IL-6 receptor antibody having a sequence of native mouse IgG1.

본 발명은 환원 조건하에서, 제1 항원 결합 활성을 갖는 폴리펩티드 및 그 제1 항원 결합 활성과 다른 제2 항원 결합 활성을 갖는 폴리펩티드의 각 호모체의 해리를 촉진하고, 헤테로체의 회합을 제어하기 위해, 중쇄 CH3영역의 아미노산 잔기를 개변함으로써 목적하는 이종 다량체를 제조하는 방법에 관한 것이다. 또한 본 발명은 목적하는 이종 다량체를 선택하는 방법에 관한 것이다. The present invention promotes the dissociation of homoforms of a polypeptide having a first antigen-binding activity and a polypeptide having a second antigen-binding activity different from the first antigen-binding activity under reducing conditions, and controls the association of heteroforms. , It relates to a method for producing a desired heteromultimer by modifying the amino acid residues of the heavy chain CH3 region. The present invention also relates to a method for selecting a desired heteromultimer.

용어의 정의Definition of Terms

본 발명에 있어서의 「폴리펩티드 」란, 아미노산 서열 중에 중쇄 Fc영역을 포함하는 폴리펩티드(Fc영역 함유 폴리펩티드) 및 단백질(Fc영역 함유 단백질)을 가리킨다. 또한 통상 생물 유래의 폴리펩티드이나 특별히 한정되지 않고, 예를 들면 인공적으로 설계된 서열로 이루어지는 폴리펩티드여도 된다. 또한 천연 폴리펩티드 또는 합성 폴리펩티드, 재조합 폴리펩티드 등 중 어느 것이어도 된다. 또한 상기 폴리펩티드의 단편도 또한 본 발명의 폴리펩티드에 포함된다. The "polypeptide" in the present invention refers to a polypeptide (Fc region-containing polypeptide) and protein (Fc region-containing protein) containing a heavy chain Fc region in an amino acid sequence. In addition, it is not particularly limited to a polypeptide derived from a living organism, but may be, for example, a polypeptide composed of an artificially designed sequence. Moreover, any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, etc. may be sufficient. In addition, fragments of the above polypeptides are also included in the polypeptides of the present invention.

본 명세서에 있어서 「항체」란, 천연의 것이거나 또는 부분적 또는 완전 합성에 의해 제조된 면역글로불린을 말한다. 항체는 그것이 천연으로 존재하는 혈장이나 혈청 등의 천연 자원이나 항체를 생산하는 하이브리도마 세포의 배양상청으로부터 단리될 수 있고, 또는 유전자 재조합 등의 수법을 사용함으로써 부분적으로 또는 완전히 합성될 수 있다. 항체의 예로서는 면역글로불린의 아이소타입 및 그들 아이소타입의 서브클래스를 적합하게 들 수 있다. 인간의 면역글로불린으로서 IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM의 9종류의 클래스(아이소타입)가 알려져 있다. 마우스의 면역글로불린으로서 IgG1, IgG2a, IgG2b, IgG3의 4종류의 클래스가 알려져 있다. 본 발명의 항체에는 이들 아이소타입 중 인간의 면역글로불린으로서는 IgG1, IgG2, IgG3, IgG4가, 마우스의 면역글로불린으로서는 IgG1, IgG2a, IgG2b, IgG3가 포함될 수 있다. 마우스의 면역글로불린으로서는 IgG1이 바람직하다. 인간 IgG1, 인간 IgG2, 인간 IgG3, 인간 IgG4 불변영역으로서는 유전자 다형에 의한 복수의 알로타입 서열이 Sequences of proteins of immunological interest, NIH Publication No.91-3242에 기재되어 있는데, 본 발명에 있어서는 그 중 어느 것이어도 된다. 특히 인간 IgG1의 서열로서는 EU 넘버링으로 표시되는 356-358번 위치의 아미노산 서열이 DEL이어도 되고 EEM이어도 된다. 또한 인간 Igκ(Kappa) 불변영역과 인간 Igλ(Lambda) 불변영역으로서는, 유전자 다형에 의한 복수의 알로타입 서열이 Sequences of proteins of immunological interest, NIH Publication No.91-3242에 기재되어 있는데, 본 발명에 있어서는 그 중 어느 것이어도 된다. As used herein, the term "antibody" refers to an immunoglobulin that is natural or partially or completely synthesized. Antibodies can be isolated from natural sources, such as plasma or serum, in which they occur naturally, or from the culture supernatant of hybridoma cells that produce the antibody, or can be partially or completely synthesized by using techniques such as genetic recombination. Suitable examples of the antibody include isotypes of immunoglobulins and subclasses of those isotypes. As human immunoglobulins, nine classes (isotypes) are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. As mouse immunoglobulins, four classes are known: IgG1, IgG2a, IgG2b, and IgG3. Among these isotypes, the antibody of the present invention may include IgG1, IgG2, IgG3, and IgG4 as human immunoglobulins, and IgG1, IgG2a, IgG2b, and IgG3 as mouse immunoglobulins. As the mouse immunoglobulin, IgG1 is preferable. As the human IgG1, human IgG2, human IgG3, and human IgG4 constant regions, a plurality of allotype sequences according to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242. In the present invention, any one of them it may be In particular, as the sequence of human IgG1, the amino acid sequence at positions 356-358 indicated by EU numbering may be DEL or EEM. In addition, as the human Igκ (Kappa) constant region and the human Igλ (Lambda) constant region, a plurality of allotype sequences due to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242, in the present invention If there is, any of them may be sufficient.

「Fc영역」이라는 용어는 천연서열 Fc영역 및 변이형 Fc영역을 포함하는 면역글로불린 중쇄의 C말단영역을 정의하기 위해 사용된다. 면역글로불린 중쇄의 Fc영역의 경계는 변화될 지도 모르지만, 항체 분자 중의 힌지부 또는 그의 일부, CH2, CH3 도메인으로 이루어지는 영역을 말한다. 통상 인간 IgG 중쇄 Fc영역은 Cys 226 위치의 아미노산 잔기로부터 Fc영역의 카르복실 말단까지 신장되는 것으로 정의되나, 이것에 한정되지 않는다. 면역글로불린의 Fc영역은 2개의 불변영역, CH2 및 CH3를 포함한다. 인간 IgG Fc영역의「CH2」도메인은 통상 아미노산 231부터 아미노산 340까지 신전된다. 「CH3」도메인은 Fc영역에 있어서의 카르복실 말단에서 CH2영역까지 신전된다. 즉, IgG의 아미노산 341부터 대략 아미노산 447까지 신전된다. The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain including a native sequence Fc region and a mutant Fc region. Although the boundary of the Fc region of an immunoglobulin heavy chain may change, it refers to a region consisting of the hinge region in an antibody molecule or a part thereof, CH2, CH3 domains. Usually, the human IgG heavy chain Fc region is defined as extending from the amino acid residue at Cys 226 to the carboxyl terminus of the Fc region, but is not limited thereto. The Fc region of immunoglobulin includes two constant regions, CH2 and CH3. The "CH2" domain of the human IgG Fc region is usually extended from amino acid 231 to amino acid 340. The "CH3" domain extends from the carboxyl terminus in the Fc region to the CH2 region. That is, it extends from amino acid 341 to approximately amino acid 447 of IgG.

Fc영역은 IgG 단일클론 항체 등을 펩신 등의 단백질 분해 효소로 부분 소화한 후에, 프로테인 A 또는 프로테인 G 칼럼에 흡착된 분획을 재용출함으로써 적합하게 취득할 수 있다. 이러한 단백질 분해 효소로서는 pH 등의 효소의 반응 조건을 적절히 설정함으로써 제한적으로 Fab나 F(ab')2를 생성시키도록 전장 항체를 소화할 수 있는 것이라면 특단의 한정은 되지 않고, 예를 들면 펩신이나 파파인 등을 예시할 수 있다.The Fc region can be suitably obtained by partially digesting an IgG monoclonal antibody or the like with a proteolytic enzyme such as pepsin and then re-eluting the fraction adsorbed to the Protein A or Protein G column. As such a proteolytic enzyme, there is no particular limitation as long as it can digest the full-length antibody to produce Fab or F(ab')2 by appropriately setting the reaction conditions of the enzyme such as pH. For example, pepsin or Papain and the like can be exemplified.

개변 개소 번호에 대해서는 EU 넘버링(Kabat EA et al． 1991． Sequences of Proteins of Immunological Interest．NIH)을 채용하였다. For the number of modified sites, EU numbering (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH) was adopted.

본 발명에 있어서의 폴리펩티드의 「회합(association)」이란, 예를 들면 2 이상의 폴리펩티드 영역이 상호작용하는 상태를 가리키는 것으로 환언할 수 있다. In the present invention, "association" of a polypeptide can be put in other words, indicating a state in which two or more polypeptide regions interact.

본 발명에 있어서 「회합을 제어한다」는 것은 목적하는 회합 상태가 되도록 제어하는 것을 말하고, 보다 구체적으로는, 폴리펩티드 간에 있어서의 바람직하지 않은 회합(바람직하게는 동일 아미노산 서열을 갖는 폴리펩티드끼리의 회합)이 형성되기 어렵게 하도록 제어하는 것을 말한다. In the present invention, "controlling the association" means controlling the desired association state, and more specifically, undesirable association between polypeptides (preferably association between polypeptides having the same amino acid sequence). It means controlling so that it is difficult to form.

본 발명에 있어서의 「계면(interface)」이란 통상 회합(상호작용)할 때의 회합면을 가리키고, 계면을 형성하는 아미노산 잔기란 통상 그 회합에 제공되는 폴리펩티드 영역에 포함되는 1 또는 복수의 아미노산 잔기로, 보다 바람직하게는 회합 시에 접근하여 상호작용에 관여하는 아미노산 잔기를 말한다. 그 상호작용에는, 구체적으로는 회합 시에 접근하는 아미노산 잔기끼리가 수소 결합, 정전적 상호작용, 가교를 형성하고 있는 경우 등이 포함된다. In the present invention, the term "interface" refers to an association surface at the time of normal association (interaction), and the amino acid residue forming the interface refers to one or more amino acid residues usually contained in the polypeptide region provided for the association. , more preferably, it refers to an amino acid residue involved in the interaction by accessing it at the time of association. Specifically, the interaction includes a case in which amino acid residues approaching at the time of association form a hydrogen bond, an electrostatic interaction, or a bridge.

본 발명에 있어서의 폴리펩티드의 「호모체」란 동일한 아미노산 서열을 갖는 폴리펩티드끼리가 회합되어 있는 상태를 말한다. The "homobody" of a polypeptide in the present invention refers to a state in which polypeptides having the same amino acid sequence are associated with each other.

본 발명에 있어서의 폴리펩티드의 「헤테로체」란 제1 폴리펩티드와, 아미노산 서열에 있어서 제1 폴리펩티드와는 1개 이상의 아미노산 잔기가 다른 제2 폴리펩티드가 회합되어 있는 상태를 말한다. The "heteroform" of a polypeptide in the present invention refers to a state in which a first polypeptide and a second polypeptide having at least one amino acid residue different from the first polypeptide in the amino acid sequence are associated with each other.

본 발명에 있어서의 폴리펩티드의 「해리」란, 폴리펩티드의 호모체에 있어서 2 이상의 폴리펩티드가 회합된 상태에서 각 폴리펩티드 단체(單體)로 분리된 상태를 말한다. The "dissociation" of a polypeptide in the present invention refers to a state in which two or more polypeptides are associated with each other in a homogeneous body of the polypeptide and separated into individual polypeptides.

본 발명에 있어서 「이종 다량체(헤테로 멀티머)」란, 복수 종의 폴리펩티드로 구성되고, 그 폴리펩티드가 서로 회합 가능한 단백질의 다량체를 말한다. 보다 상세하게는 「이종 다량체」는 적어도 제1 폴리펩티드 및 제2 폴리펩티드를 가지며, 여기에 있어서 제2 폴리펩티드는 아미노산 서열에 있어서 제1 폴리펩티드와는 1개 이상의 아미노산 잔기가 다른 분자이다. 또한 특별히 한정되지 않지만, 상기 이종 다량체는 2종 이상의 다른 리간드, 항원, 수용체, 또는 기질 등에 대해 항원 결합 활성을 갖는 것이 바람직하다. 상기 이종 다량체는 제1 및 제2 폴리펩티드에 의해 형성되는 「이종 이량체」에 더하여, 추가로 다른 종의 폴리펩티드가 존재하고 있어도 된다. 즉, 본 발명의 「이종 다량체」는 이종 이량체에 제한되지 않고, 예를 들면 이종 3량체, 이종 4량체 등도 포함된다. In the present invention, the term "heteromultimer (heteromultimer)" refers to a protein multimer composed of a plurality of types of polypeptides and the polypeptides can associate with each other. More specifically, a "heteromultimer" has at least a first polypeptide and a second polypeptide, wherein the second polypeptide is a molecule different from the first polypeptide by one or more amino acid residues in the amino acid sequence. Further, although not particularly limited, the heteromultimer preferably has antigen-binding activity against two or more different ligands, antigens, receptors, substrates, and the like. In addition to the "heterodimer" formed by the first and second polypeptides, the heteromultimer may further include a polypeptide of another species. That is, the "heteromultimer" of the present invention is not limited to a heterodimer, and includes, for example, a heterotrimer, a heterotetramer, and the like.

본 발명의 제1 폴리펩티드, 제2 폴리펩티드 및 1개 또는 2개의 제3 폴리펩티드를 포함하는 폴리펩티드 다량체에 있어서, 제1 폴리펩티드 및 제2 폴리펩티드는 각각 제3 폴리펩티드와 다량체(이량체)를 형성할 수 있다. 또한 형성된 이량체끼리가 다량체(4량체)를 형성할 수 있다. 2개의 제3 폴리펩티드는 완전히 동일한 아미노산 서열을 가지고 있어도 된다(동일한 항원에 대해 결합 활성을 가지고 있어도 된다). 또는 동일한 아미노산 서열이면서 2 이상의 활성이 있어도 된다(예를 들면 2 이상의 다른 항원에 대해 결합 활성을 가지고 있어도 된다). 또한 제3 폴리펩티드가 1개인 경우는, 제3 폴리펩티드는 제1 폴리펩티드 또는 제2 폴리펩티드 중 어느 한쪽과 이량체를 형성하여 폴리펩티드 다량체를 형성할 수 있다. In a polypeptide multimer comprising a first polypeptide, a second polypeptide and one or two third polypeptides of the invention, the first polypeptide and the second polypeptide are each capable of forming a multimer (dimer) with the third polypeptide. can Moreover, the formed dimers can form a multimer (tetramer). The two third polypeptides may have the completely identical amino acid sequence (they may have binding activity to the same antigen). Alternatively, the same amino acid sequence may have two or more activities (for example, it may have binding activity to two or more different antigens). In addition, when there is only one third polypeptide, the third polypeptide can form a dimer with either the first polypeptide or the second polypeptide to form a polypeptide multimer.

본 발명의 폴리펩티드 다량체에 있어서, 제1 폴리펩티드와 제2 폴리펩티드는 다른 항원에 대해 결합 활성을 갖는 것이 바람직하다. 한편 제3 폴리펩티드는 제1 폴리펩티드나 제2 폴리펩티드 중 어느 하나 또는 양쪽과 동일한 항원에 대해 결합 활성을 갖는 폴리펩티드여도 된다. 또는 제1 폴리펩티드나 제2 폴리펩티드와는 다른 항원에 대해 결합 활성을 갖는 폴리펩티드여도 된다.In the polypeptide multimer of the present invention, it is preferable that the first polypeptide and the second polypeptide have binding activity to different antigens. On the other hand, the third polypeptide may be a polypeptide having the same antigen-binding activity as either or both of the first polypeptide and the second polypeptide. Alternatively, it may be a polypeptide having an antigen-binding activity different from that of the first polypeptide or the second polypeptide.

또는 본 발명의 폴리펩티드 다량체는 제1 폴리펩티드, 제2 폴리펩티드, 제3 폴리펩티드 및 제4 폴리펩티드를 포함하는 폴리펩티드 다량체로 하는 것도 가능하다. 이러한 폴리펩티드 다량체에 있어서, 제1 폴리펩티드 및 제2 폴리펩티드는 각각 제3 폴리펩티드 및 제4 폴리펩티드와 다량체(이량체)를 형성할 수 있다. 예를 들면 제1 폴리펩티드와 제3 폴리펩티드, 제2 폴리펩티드와 제4 폴리펩티드 사이에서 디설피드 결합을 형성함으로써 이량체를 형성할 수 있다. Alternatively, the polypeptide multimer of the present invention may be a polypeptide multimer comprising a first polypeptide, a second polypeptide, a third polypeptide and a fourth polypeptide. In such a polypeptide multimer, the first polypeptide and the second polypeptide may form a multimer (dimer) with the third and fourth polypeptides, respectively. For example, dimers can be formed by forming disulfide bonds between the first and third polypeptides, and between the second and fourth polypeptides.

본 발명의 폴리펩티드 다량체에 있어서는, 제1 폴리펩티드와 제2 폴리펩티드는 다른 항원에 대해 결합 활성을 갖는 것이 바람직하다. 한편 제3 폴리펩티드는 제1 폴리펩티드나 제2 폴리펩티드 중 어느 하나 또는 양쪽과 동일한 항원에 대해 결합 활성을 갖는 폴리펩티드여도 된다. 또는 제1 폴리펩티드나 제2 폴리펩티드와는 다른 항원에 대해 결합 활성을 갖는 폴리펩티드여도 된다. 또한 제4 폴리펩티드는 제1 폴리펩티드나 제2 폴리펩티드 중 어느 하나와 동일한 항원에 대해 결합 활성을 갖는 폴리펩티드여도 된다. 또는 제1 폴리펩티드나 제2 폴리펩티드와는 다른 항원에 대해 결합 활성을 갖는 폴리펩티드여도 된다. In the polypeptide multimer of the present invention, it is preferable that the first polypeptide and the second polypeptide have binding activity to different antigens. On the other hand, the third polypeptide may be a polypeptide having the same antigen-binding activity as either or both of the first polypeptide and the second polypeptide. Alternatively, it may be a polypeptide having an antigen-binding activity different from that of the first polypeptide or the second polypeptide. Further, the fourth polypeptide may be a polypeptide having the same antigen-binding activity as either the first polypeptide or the second polypeptide. Alternatively, it may be a polypeptide having an antigen-binding activity different from that of the first polypeptide or the second polypeptide.

본 발명에 있어서의 「이종 다량체」가 이중특이성 항체일 때, 예를 들면 제1 폴리펩티드와 제2 폴리펩티드가 각각 항원 A에 대한 항체 중쇄의 아미노산 서열을 포함하는 폴리펩티드, 항원 B에 대한 항체 중쇄의 아미노산 서열을 포함하는 폴리펩티드인 경우, 제3 폴리펩티드를 항원 A에 대한 항체 경쇄의 아미노산 서열을 포함하는 폴리펩티드, 제4 폴리펩티드를 항원 B에 대한 항체 경쇄의 아미노산 서열을 포함하는 폴리펩티드로 할 수 있다. When the "heteromultimer" in the present invention is a bispecific antibody, for example, the first polypeptide and the second polypeptide each contain the amino acid sequence of the antibody heavy chain against antigen A, the heavy chain of the antibody against antigen B In the case of a polypeptide comprising an amino acid sequence, the third polypeptide may be a polypeptide comprising the amino acid sequence of an antibody light chain directed against antigen A, and the fourth polypeptide may be a polypeptide comprising the amino acid sequence of an antibody light chain directed against antigen B.

본 발명에 있어서 「항원 결합 활성을 갖는 폴리펩티드」란, 항체 중쇄 또는 경쇄의 가변영역, 수용체, 수용체와 Fc영역의 융합 펩티드, 스캐폴드(Scaffold) 및 이들의 단편 등, 항원, 리간드 등의 단백질이나 펩티드에 대해 결합 가능한 도메인(영역)을 갖는 5 아미노산 이상의 길이를 갖는 펩티드 및 단백질을 가리킨다. 즉 항원 결합 활성을 갖는 폴리펩티드는 항체 가변영역, 수용체, 수용체와 Fc영역의 융합 펩티드, 스캐폴드 또는 이들 단편의 아미노산 서열을 포함할 수 있다. In the present invention, "polypeptide having antigen-binding activity" means an antibody heavy or light chain variable region, receptor, receptor and Fc region fusion peptides, scaffolds and fragments thereof, proteins such as antigens, ligands, etc. Refers to peptides and proteins having a length of 5 amino acids or more having a domain (region) capable of binding to the peptide. That is, the polypeptide having antigen-binding activity may include an amino acid sequence of an antibody variable region, a receptor, a fusion peptide of a receptor and an Fc region, a scaffold, or a fragment thereof.

스캐폴드로서는 하나 이상의 항원에 결합할 수 있는 입체 구조적으로 안정한 폴리펩티드라면 어떠한 폴리펩티드라도 사용할 수 있다. 이러한 폴리펩티드로서는, 예를 들면 항체 가변영역 단편, 피브로넥틴, Protein A 도메인, LDL 수용체 A 도메인, 리포칼린 등 외에, Nygren 등(Current Opinion in Structural Biology, 7:463-469(1997), Journal of Immunol Methods, 290:3-28(2004)), Binz 등(Nature Biotech 23:1257-1266(2005)), Hosse 등(Protein Science 15:14-27(2006))에 기재된 분자를 들 수 있는데 이들에 한정되지 않는다. As a scaffold, any polypeptide can be used as long as it is a conformationally stable polypeptide capable of binding to one or more antigens. Examples of such polypeptides include antibody variable region fragments, fibronectin, Protein A domain, LDL receptor A domain, lipocalin, etc., and Nygren et al. (Current Opinion in Structural Biology, 7:463-469 (1997), Journal of Immunol Methods). , 290:3-28 (2004)), Binz et al. (Nature Biotech 23:1257-1266 (2005)), and Hosse et al. (Protein Science 15:14-27 (2006)). doesn't happen

항체의 가변영역, 수용체, 수용체와 Fc영역의 융합 펩티드, 스캐폴드 및 이들 단편의 취득방법은 당업자에게 주지이다. 영역의 아미노산 서열과 항체 경쇄 불변영역의 아미노산 서열을 포함하는 폴리펩티드를 사용하는 것도 가능하다. Methods for obtaining antibody variable regions, receptors, receptor-Fc region fusion peptides, scaffolds, and fragments thereof are well known to those skilled in the art. It is also possible to use a polypeptide comprising the amino acid sequence of the region and the amino acid sequence of the antibody light chain constant region.

본 발명에 있어서의 「환원 조건」이란, 중쇄 힌지영역 내의 중쇄 간 디설피드 결합을 형성하는 시스테인 잔기가 산화되는 보다 환원된 상태가 될 가능성이 높은 조건 또는 환경을 가리키고, 바람직하게는 중쇄 간 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키는 것을 가능하게 하는 조건 또는 환경을 가리키며, 특히 바람직하게는 힌지영역 외의 시스테인은 디설피드 결합의 유의한 이성화를 일으키지 않으나(즉, 중쇄와 경쇄 간의 디설피드 결합을 보존한 채), 중쇄 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키는 것을 가능하게 하는 조건 또는 환경을 가리킨다. 예를 들면 본 발명에 있어서，환원 조건하에서 Fc영역을 포함하는 제1 폴리펩티드의 호모와 Fc영역을 포함하는 제2 폴리펩티드를 함께 인큐베이트하는 시간은 당업자가 적절히 설정할 수 있다. The "reducing conditions" in the present invention refer to conditions or environments in which the cysteine residue forming the inter-heavy chain disulfide bond in the heavy chain hinge region is highly likely to be in a reduced state, preferably the inter-heavy chain hinge region. Refers to conditions or circumstances that allow cysteines within the disulfide bond to cause isomerization of disulfide bonds, particularly preferably cysteines outside the hinge region do not cause significant isomerization of disulfide bonds (i.e., disulfide bonds between heavy and light chains) conserved), refers to a condition or environment that enables cysteine in the heavy chain hinge region to cause isomerization of a disulfide bond. For example, in the present invention, the time for incubating the homo of the first polypeptide comprising the Fc region and the second polypeptide comprising the Fc region under reducing conditions can be appropriately set by those skilled in the art.

본 발명에 있어서의 「환원제」란, 그 환경에 있어서 분자를 환원하는 화합물, 즉 그 환경에 있어서 분자를 보다 환원환 상태로 및 보다 환원하고 있는 상태로 바꾸는 화합물을 가리킨다. 환원제는 전자를 공여함으로써 작용하고, 그로 인해 기질의 환원 후에 그 자체는 산화된 상태가 된다. 따라서, 환원제란 전자를 공여하는 작용물질이다. 환원제의 예로는, 디티오트레이톨(DTT), 메르캅토에탄올, 시스테인, 티오글리콜산, 시스테아민(2-메르캅토에틸아민：2-MEA), 글루타티온(GSH), TCEP(트리스(2-카르복시에틸)포스핀) 및 수소화붕소나트륨이 포함된다. The "reducing agent" in the present invention refers to a compound that reduces a molecule in its environment, that is, a compound that converts a molecule to a more reduced ring state and a more reduced state in the environment. The reducing agent acts by donating electrons so that, after reduction of the substrate, it itself is in an oxidized state. Thus, a reducing agent is an electron-donating agent. Examples of the reducing agent include dithiothreitol (DTT), mercaptoethanol, cysteine, thioglycolic acid, cysteamine (2-mercaptoethylamine: 2-MEA), glutathione (GSH), TCEP (tris (2- carboxyethyl)phosphine) and sodium borohydride.

본 발명에 있어서의 「중쇄 간 디설피드 결합의 이성화」란, 다른 중쇄에 포함되는 시스테인 간에서의 디설피드 결합의 교환, 즉 디설피드 결합의 재편성을 가리킨다. "Isomerization of disulfide bond between heavy chains" in the present invention refers to exchange of disulfide bonds between cysteines contained in different heavy chains, ie, reorganization of disulfide bonds.

「디설피드 결합 형성」이란, 1개 또는 2개의 폴리펩티드 중에 존재하는 2개의 시스테인 간에서 공유 결합을 형성하는 과정을 가리키고, 이 결합은 「-S--S-」로서 도시화된다."Disulfide bond formation" refers to the process of forming a covalent bond between two cysteines present in one or two polypeptides, and this bond is illustrated as "-S--S-".

「디설피드 결합의 환원」이란, 디설피드 결합의 절단에 의해 2개의 티올기(-SH기)가 생성되는 과정을 가리킨다. "Reduction of a disulfide bond" refers to a process in which two thiol groups (-SH groups) are produced by cleavage of a disulfide bond.

본 발명에 있어서의 「FcγR」또는「FcgR」이란 Fcγ 수용체로, IgG1, IgG2, IgG3, IgG4 단일클론 항체의 Fc영역에 결합 가능한 수용체를 말하고, 실질적으로 Fcγ 수용체 유전자에 코드되는 단백질 패밀리의 모든 멤버를 의미한다. 인간의 경우는 이 패밀리에는, 예를 들면 아이소폼 FcγRIa, FcγRIb 및 FcγRIc를 포함하는 FcγRI(CD64)；아이소폼 FcγRIIa(알로타입 H131(H형) 및 R131(R형)을 포함), FcγRIIb(FcγRIIb-1 및 FcγRIIb-2를 포함) 및 FcγRIIc를 포함하는 FcγRII(CD32)； 및 아이소폼 FcγRIIIa(알로타입 V158 및 F158을 포함) 및 FcγRIIIb(알로타입 FcγRIIIb-NA1 및 FcγRIIIb-NA2를 포함)를 포함하는 FcγRIII(CD16) 및 어떠한 미발견의 인간 FcγR류 또는 FcγR 아이소폼 또는 알로타입도 포함된다. FcγR은 인간, 마우스, 랫트, 토끼 및 원숭이 유래의 것이 포함되는데 이들에 한정되지 않고, 어떠한 생물 유래여도 된다. 마우스 FcγR류에는, 예를 들면 FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16) 및 FcγRIII-2(CD16-2), FcγRIV 및 어떠한 미발견의 마우스 FcγR류 또는 FcγR 아이소폼 또는 알로타입도 포함된다. In the present invention, "FcγR" or "FcgR" is an Fcγ receptor, refers to a receptor capable of binding to the Fc region of an IgG1, IgG2, IgG3, IgG4 monoclonal antibody, substantially all members of the protein family encoded by the Fcγ receptor gene. means In the case of humans, this family includes, for example, FcγRI (CD64) containing isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) including -1 and FcγRIIb-2) and FcγRIIc; and isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) FcγRIII (CD16) and any undiscovered human FcγRs or FcγR isoforms or allotypes are included. FcγR includes those derived from humans, mice, rats, rabbits and monkeys, but is not limited thereto, and may be derived from any organism. Mouse FcγRs include, for example, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), FcγRIV and any undiscovered mouse FcγRs or FcγR isoforms or allotypes. do.

아미노산 잔기의 전하 반발을 이용한 개변에 의한 이종 다량체의 제조방법Method for producing heteromultimers by modification using charge repulsion of amino acid residues

상기 방법의 바람직한 태양에 있어서는, 본 발명은 2종 이상의 다량체를 형성할 수 있는 이종 다량체에 대해서, 제1 및 제2 폴리펩티드의 호모체의 해리를 촉진하여 1종 이상의 다량체를 형성하는 폴리펩티드 간의 회합을 제어하기 위해, 폴리펩티드 간의 계면을 형성하는 아미노산 잔기를 개변하여 목적하는 폴리펩티드의 헤테로체를 제조하는 방법이다.In a preferred aspect of the method, the present invention provides a polypeptide that promotes dissociation of homomers of the first and second polypeptides to form one or more multimers with respect to a heteromultimer capable of forming two or more types of multimers In order to control the association between the polypeptides, it is a method for producing a heteroform of a desired polypeptide by modifying the amino acid residues forming the interface between the polypeptides.

또한 본 발명의 제1 항원 결합 활성을 갖는 폴리펩티드 및 제2 항원 결합 활성을 갖는 폴리펩티드는, 항체 중쇄 불변영역의 아미노산 서열 또는 항체 Fc영역의 아미노산 서열을 포함할 수 있다. 항체 Fc영역 또는 항체 중쇄 불변영역의 아미노산 서열로서는, 인간 IgG 타입의 불변영역 또는 Fc영역의 아미노산 서열을 들 수 있는데 이들에 한정되지 않는다. IgG 타입의 불변영역 또는 Fc영역으로서는, 천연형 IgG1, IgG2, IgG3, IgG4의 아이소타입 중 어느 것이어도 된다. 또는 이들의 개변체여도 된다. Fc영역의 EU 넘버링 447번 위치의 리신 및 EU 넘버링 446번 위치의 글리신이 이들을 코드하는 핵산을 재조합 유전자 조작함으로써 제거되어도 된다. In addition, the polypeptide having a first antigen-binding activity and a polypeptide having a second antigen-binding activity of the present invention may include an amino acid sequence of an antibody heavy chain constant region or an amino acid sequence of an antibody Fc region. Examples of the amino acid sequence of the antibody Fc region or antibody heavy chain constant region include, but are not limited to, human IgG-type constant region or Fc region amino acid sequence. As the constant region or Fc region of the IgG type, any of the isotypes of native IgG1, IgG2, IgG3, and IgG4 may be used. Or these modified bodies may be sufficient. Lysine at EU numbering position 447 and glycine at EU numbering position 446 of the Fc region may be removed by recombinantly engineering a nucleic acid encoding them.

또한 본 발명의 제3 항원 결합 활성을 갖는 폴리펩티드 및 제4 항원 결합 활성을 갖는 폴리펩티드는, 항체 경쇄 불변영역의 아미노산 서열을 포함할 수 있다. 항체 경쇄 불변영역의 아미노산 서열로서는 인간 kappa, 인간 lambda 타입의 불변영역의 아미노산 서열을 들 수 있는데 이들에 한정되지 않는다. 또는 이들의 개변체여도 된다. In addition, the polypeptide having the third antigen-binding activity and the polypeptide having the fourth antigen-binding activity of the present invention may include an amino acid sequence of an antibody light chain constant region. Examples of the amino acid sequence of the antibody light chain constant region include, but are not limited to, the amino acid sequence of the constant region of human kappa and human lambda type. Or these modified bodies may be sufficient.

또한 본 발명의 항원 결합 활성을 갖는 폴리펩티드는, 항체 가변영역의 아미노산 서열(예를 들면 CDR1, CDR2, CDR3, FR1, FR2, FR3, FR4의 아미노산 서열)을 포함할 수 있다. In addition, the polypeptide having antigen-binding activity of the present invention may include the amino acid sequence of an antibody variable region (eg, the amino acid sequence of CDR1, CDR2, CDR3, FR1, FR2, FR3, FR4).

또한 본 발명의 폴리펩티드 간의 해리 및/또는 회합 제어방법의 바람직한 태양에 있어서는, 당해 방법으로서 중쇄의 불변영역의 계면에 전하적인 반발을 도입하여 중쇄끼리의 회합을 억제시키는 방법을 들 수 있다. 중쇄 불변영역의 계면에서 접촉하는 아미노산 잔기로서는, 예를 들면 CH3영역에 있어서의 356번 위치와 439번 위치, 357번 위치와 370번 위치, 399번 위치와 409번 위치에 상당하는 영역을 들 수 있다. 중쇄 불변영역에 대해서는 EU 넘버링에 의해 표시하였다. In a preferred embodiment of the method for controlling dissociation and/or association between polypeptides of the present invention, the method includes a method of inhibiting association of heavy chains by introducing an electric charge repulsion at the interface of the constant region of the heavy chains. Examples of the amino acid residues in contact with the heavy chain constant region interface include regions corresponding to positions 356 and 439, 357 and 370, 399 and 409 in the CH3 region. have. The heavy chain constant region was indicated by EU numbering.

후술하는 실시예에서 나타내는 바와 같이, 이들 아미노산 잔기를 개변하고 본 발명의 방법을 실시함으로써, 폴리펩티드 간의 중쇄의 해리 및/또는 회합을 제어하여 목적하는 이종 다량체를 우선적으로 취득할 수 있다. 즉, 바람직한 태양에 있어서 본 발명은 2종 이상의 중쇄 Fc영역을 포함하는 항체 및 Fc영역 함유 단백질(예를 들면 IgG형 항체, minibody(Alt M et al. FEBS Letters 199 9; 454: 90-94), 면역어드헤신(비특허문헌 2) 등)로서, 제1 중쇄 Fc영역에 있어서의 아래의 (1)~(3)에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동종의 전하를 갖는 폴리펩티드를 제공한다. As shown in Examples to be described later, by modifying these amino acid residues and carrying out the method of the present invention, the desired heteromultimer can be preferentially obtained by controlling the dissociation and/or association of heavy chains between polypeptides. That is, in a preferred embodiment, the present invention provides an antibody comprising two or more heavy chain Fc regions and an Fc region-containing protein (eg, an IgG-type antibody, minibody (Alt M et al. FEBS Letters 1999 9; 454: 90-94) , immunoadhesins (non-patent document 2, etc.), 1 to 3 sets of amino acid residues selected from the group of amino acid residues shown in the following (1) to (3) in the first heavy chain Fc region are the same A polypeptide having a charge is provided.

(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기(1) amino acid residues at positions 356 and 439 by EU numbering

(2) EU 넘버링에 의한 357번 위치 및 370번 위치의 아미노산 잔기(2) amino acid residues at positions 357 and 370 by EU numbering

(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기(3) amino acid residues at positions 399 and 409 by EU numbering

또한 본 발명은 상기 제1 중쇄 Fc영역과는 다른 제2 중쇄 Fc영역에 있어서의 상기 (1)~(3)에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기로서, 상기 제1 중쇄 Fc영역에 있어서의 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 동종의 전하를 갖는 조와 동일한 위치의 아미노산 잔기가 상기 제1 중쇄 Fc영역에 있어서의 아미노산 잔기와는 반대의 전하를 갖는 폴리펩티드를 제공한다. In addition, the present invention is a set of 1 to 3 amino acid residues selected from the group of amino acid residues shown in (1) to (3) above in a second heavy chain Fc region different from the first heavy chain Fc region, In the group of amino acid residues shown in (1) to (3) in the heavy chain Fc region, the amino acid residue at the same position as the group having the same charge has a charge opposite to that of the amino acid residue in the first heavy chain Fc region It provides a polypeptide having.

상기 폴리펩티드에 있어서 「전하를 갖는 아미노산 잔기」는, 예를 들면 아래의 (a) 또는 (b) 중 어느 하나의 군에 포함되는 아미노산 잔기로부터 선택되는 것이 바람직하다.The "amino acid residue having a charge" in the polypeptide is preferably selected from, for example, amino acid residues included in any one of the following groups (a) and (b).

(a) 글루타민산(E), 아스파라긴산(D);(a) glutamic acid (E), aspartic acid (D);

(b) 리신(K), 아르기닌(R), 히스티딘(H). (b) Lysine (K), Arginine (R), Histidine (H).

상기 폴리펩티드에 있어서 「동종의 전하를 갖는」다는 것은, 예를 들면 2개 이상의 아미노산 잔기 모두가 상기 (a) 또는 (b) 중 어느 하나의 군에 포함되는 아미노산 잔기를 갖는 것을 의미한다. 「반대의 전하를 갖는」다는 것은, 예를 들면 2개 이상의 아미노산 잔기 중 하나 이상의 아미노산 잔기가 상기 (a) 또는 (b) 중 어느 하나의 군에 포함되는 아미노산 잔기를 갖는 경우에, 나머지 아미노산 잔기가 다른 군에 포함되는 아미노산 잔기를 갖는 것을 의미한다. In the polypeptide, "having the same charge" means, for example, that all two or more amino acid residues have an amino acid residue included in any one of the groups (a) or (b). "Having an opposite charge" means, for example, when one or more amino acid residues among two or more amino acid residues have an amino acid residue included in either group of (a) or (b), the remaining amino acid residues means having an amino acid residue included in another group.

바람직한 태양에 있어서, 상기 폴리펩티드는 제1 중쇄 CH3영역과 제2 중쇄 CH3영역이 디설피드 결합에 의해 가교되어 있어도 된다. In a preferred embodiment, in the polypeptide, the first heavy chain CH3 region and the second heavy chain CH3 region may be crosslinked by a disulfide bond.

본 발명에 있어서의 「회합 계면 제어 개변」으로서, 예를 들면 아래의 개변을 들 수 있다：As the "association interface control modification" in the present invention, for example, the following modifications are mentioned:

(1) 제1 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 356번 위치의 Asp(D)의 Lys(K), Arg(R) 또는 His(H)로의 개변, 및 제2 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 439번 위치의 Lys(K)의 Glu(E) 또는 Asp(D)로의 개변；(1) Lys (K), Arg (R) or His (H) of Asp (D) at position 356 by EU numbering in the first heavy chain Fc region, and in the second heavy chain Fc region Modification of Lys (K) at position 439 by EU numbering to Glu (E) or Asp (D);

(2) 제1 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 357번 위치의 Glu(E)의 Lys(K), Arg(R) 또는 His(H)로의 개변, 및 제2 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 370번 위치의 Lys(K)의 Glu(E) 또는 Asp(D)로의 개변；(2) Glu (E) at position 357 by EU numbering in the first heavy chain Fc region to Lys (K), Arg (R) or His (H), and in the second heavy chain Fc region Alteration of Lys (K) at position 370 by EU numbering to Glu (E) or Asp (D);

(3) 제1 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 399번 위치의 Asp(D)의 Lys(K), Arg(R) 또는 His(H)로의 개변, 및 제2 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 409번 위치의 Lys(K)의 Glu(E) 또는 Asp(D)로의 개변.(3) Asp (D) at position 399 by EU numbering in the first heavy chain Fc region to Lys (K), Arg (R) or His (H), and in the second heavy chain Fc region Alteration of Lys (K) at position 409 by EU numbering to Glu (E) or Asp (D).

본 발명의 비한정의 일태양으로서 마우스 이종 다량체의 제조방법에 관한 폴리펩티드 간의 해리 및/또는 회합 제어방법의 바람직한 태양에 있어서는, 당해 방법으로서 중쇄의 불변영역의 계면에 전하적인 반발을 도입하여 중쇄끼리의 회합을 억제시키는 방법을 들 수 있다. 당해 방법에 있어서는, 중쇄 불변영역의 계면에서 접촉하는 아미노산 잔기로서는, 예를 들면 CH3영역에 있어서의 356번 위치와 439번 위치, 360번 위치와 371번 위치, 399번 위치와 409번 위치에 상당하는 영역을 들 수 있다. 중쇄 불변영역에 대해서는 EU 넘버링에 의해 표시하였다. As a non-limiting aspect of the present invention, in a preferred aspect of a method for controlling dissociation and/or association between polypeptides related to a method for producing a mouse heteromultimer, as the method, charge repulsion is introduced at the interface of the constant region of the heavy chain to introduce a heavy chain A method of suppressing the association of each other is mentioned. In this method, the amino acid residues in contact at the interface of the heavy chain constant region correspond to, for example, positions 356, 439, 360, 371, 399 and 409 in the CH3 region. area can be included. The heavy chain constant region was indicated by EU numbering.

후술하는 실시예에서 나타내는 바와 같이, 이들 마우스 유래의 CH3영역에 있어서의 아미노산 잔기를 개변하고 본 발명의 방법을 실시함으로써, 폴리펩티드 간의 중쇄의 해리 및/또는 회합을 제어하여 목적하는 이종 다량체를 우선적으로 취득할 수 있다. 즉, 바람직한 태양에 있어서, 본 발명은 2종 이상의 중쇄 Fc영역을 포함하는 항체 및 Fc영역 함유 단백질(예를 들면 IgG형 항체, minibody(Alt M et al. FEBS Letters 199 9; 454: 90-94), 면역어드헤신(비특허문헌 2) 등)로서, 제1 중쇄 Fc영역에 있어서의 아래의 (1)~(3)에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동종의 전하를 갖는 폴리펩티드를 제공한다. As shown in the Examples to be described later, by modifying the amino acid residues in the CH3 region derived from these mice and carrying out the method of the present invention, the dissociation and/or association of heavy chains between polypeptides is controlled to preferentially select the desired heteromultimer. can be obtained with That is, in a preferred embodiment, the present invention provides an antibody comprising two or more heavy chain Fc regions and an Fc region-containing protein (eg, an IgG-type antibody, a minibody (Alt M et al. FEBS Letters 1999 9; 454: 90-94). ), immunoadhesins (non-patent document 2, etc.), 1 to 3 sets of amino acid residues selected from the group of amino acid residues shown in (1) to (3) below in the first heavy chain Fc region are the same Provided is a polypeptide having a charge of

(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기(1) amino acid residues at positions 356 and 439 by EU numbering

(2) EU 넘버링에 의한 360번 위치 및 371번 위치의 아미노산 잔기(2) amino acid residues at positions 360 and 371 by EU numbering

(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기(3) amino acid residues at positions 399 and 409 by EU numbering

또한 본 발명은 상기 제1 중쇄 Fc영역과는 다른 제2 중쇄 Fc영역에 있어서의 상기 (1)~(3)에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기로서, 상기 제1 중쇄 Fc영역에 있어서의 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 동종의 전하를 갖는 조와 동일한 위치의 아미노산 잔기가, 상기 제1 중쇄 Fc영역에 있어서의 아미노산 잔기와는 반대의 전하를 갖는 폴리펩티드를 제공한다.In addition, the present invention is a set of 1 to 3 amino acid residues selected from the group of amino acid residues shown in (1) to (3) above in a second heavy chain Fc region different from the first heavy chain Fc region, In the group of amino acid residues shown in (1) to (3) in the heavy chain Fc region, the amino acid residue at the same position as the group having the same charge is the opposite charge to the amino acid residue in the first heavy chain Fc region It provides a polypeptide having a.

상기 폴리펩티드에 있어서 「전하를 갖는 아미노산 잔기」는, 예를 들면 아래의 (a) 또는 (b) 중 어느 하나의 군에 포함되는 아미노산 잔기로부터 선택되는 것이 바람직하다. The "amino acid residue having a charge" in the polypeptide is preferably selected from, for example, amino acid residues included in any one of the following groups (a) and (b).

(a) 글루타민산(E), 아스파라긴산(D)； (a) glutamic acid (E), aspartic acid (D);

(b) 리신(K), 아르기닌(R), 히스티딘(H). (b) Lysine (K), Arginine (R), Histidine (H).

상기 폴리펩티드에 있어서 「동종의 전하를 갖는」다는 것은, 예를 들면 2개 이상의 아미노산 잔기 모두가 상기 (a) 또는 (b) 중 어느 하나의 군에 포함되는 아미노산 잔기를 갖는 것을 의미한다. 「반대의 전하를 갖는」다는 것은, 예를 들면 2개 이상의 아미노산 잔기 중 하나 이상의 아미노산 잔기가 상기 (a) 또는 (b) 중 어느 하나의 군에 포함되는 아미노산 잔기를 갖는 경우에, 나머지 아미노산 잔기가 다른 군에 포함되는 아미노산 잔기를 갖는 것을 의미한다. In the polypeptide, "having the same charge" means, for example, that all two or more amino acid residues have an amino acid residue included in any one of the groups (a) or (b). "Having an opposite charge" means, for example, when one or more amino acid residues among two or more amino acid residues have an amino acid residue included in either group of (a) or (b), the remaining amino acid residues means having an amino acid residue included in another group.

바람직한 태양에 있어서, 상기 폴리펩티드는 제1 중쇄 CH3영역과 제2 중쇄 CH3영역이 디설피드 결합에 의해 가교되어 있어도 된다. In a preferred embodiment, in the polypeptide, the first heavy chain CH3 region and the second heavy chain CH3 region may be crosslinked by a disulfide bond.

본 발명에 있어서의 「회합 계면 제어 개변」으로서, 예를 들면 아래의 개변을 들 수 있다：As the "association interface control modification" in the present invention, for example, the following modifications are mentioned:

(1) 제1 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 356번 위치의 Asp(D)의 Lys(K), Arg(R) 또는 His(H)로의 개변, 및 제2 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 439번 위치의 Lys(K)의 Glu(E) 또는 Asp(D)로의 개변；(1) Lys (K), Arg (R) or His (H) of Asp (D) at position 356 by EU numbering in the first heavy chain Fc region, and in the second heavy chain Fc region Modification of Lys (K) at position 439 by EU numbering to Glu (E) or Asp (D);

(2) 제1 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 360번 위치의 Glu(E)의 Lys(K), Arg(R) 또는 His(H)로의 개변, 및 제2 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 371번 위치의 Lys(K)의 Glu(E) 또는 Asp(D)로의 개변；(2) Glu (E) at position 360 by EU numbering in the first heavy chain Fc region to Lys (K), Arg (R) or His (H), and in the second heavy chain Fc region Alteration of Lys (K) at position 371 by EU numbering to Glu (E) or Asp (D);

(3) 제1 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 399번 위치의 Asp(D)의 Lys(K), Arg(R) 또는 His(H)로의 개변, 및 제2 중쇄 Fc영역에 있어서의 EU 넘버링에 의한 409번 위치의 Lys(K)의 Glu(E) 또는 Asp(D)로의 개변(3) Asp (D) at position 399 by EU numbering in the first heavy chain Fc region to Lys (K), Arg (R) or His (H), and in the second heavy chain Fc region Modification of Lys (K) at position 409 by EU numbering to Glu (E) or Asp (D)

본 발명에 있어서 「개변」에 제공하는 아미노산 잔기로서는, 폴리펩티드의 불변영역의 아미노산 잔기에 한정되지 않는다. 당업자라면 폴리펩티드 변이체 또는 이종 다량체에 대해서 시판의 소프트웨어를 사용한 호모로지 모델링 등에 의해 계면을 형성하는 아미노산 잔기를 발견할 수 있고, 회합을 제어하도록 해당 부위의 아미노산 잔기를 개변에 제공하는 것이 가능하다. The amino acid residue to be used for "modification" in the present invention is not limited to the amino acid residue in the constant region of the polypeptide. Those skilled in the art can discover amino acid residues that form an interface for polypeptide variants or heteromultimers by homology modeling using commercially available software, etc., and it is possible to provide amino acid residues at the site for modification to control association.

본 발명의 방법에 있어서의 아미노산 잔기의 「개변」이란, 구체적으로는 원래의 아미노산 잔기를 다른 아미노산 잔기로 치환하는 것, 원래의 아미노산 잔기를 결실시키는 것, 새로운 아미노산 잔기를 부가하는 것 등을 가리키는데, 바람직하게는 원래의 아미노산 잔기를 다른 아미노산 잔기로 치환하는 것을 가리킨다. "Modification" of an amino acid residue in the method of the present invention specifically refers to replacing the original amino acid residue with another amino acid residue, deleting the original amino acid residue, adding a new amino acid residue, etc. , preferably refers to the replacement of the original amino acid residue with another amino acid residue.

397번 위치 및/또는 392번 위치의 아미노산 개변에 의한 이종 다량체의 제조방법Method for preparing a heteromultimer by amino acid modification at position 397 and/or position 392

본 발명의 폴리펩티드 간의 해리 및/또는 회합 제어방방법의 더욱 바람직한 태양에 있어서는, 당해 방법은 중쇄 CH3영역의 안정성이 불안정해지도록, 중쇄 Fc영역에 아미노산 잔기의 변이를 도입하는 것을 특징으로 하는 방법이다. 당해 방법은 임의로 상기 전하 반발 등을 이용한 계면 제어에 관한 아미노산 개변을 도입하는 공정을 추가로 포함해도 된다. In a more preferred aspect of the method for controlling dissociation and/or association between polypeptides of the present invention, the method is a method characterized in that amino acid residue mutations are introduced into the heavy chain Fc region so that the stability of the heavy chain CH3 region becomes unstable. . The method may optionally further include a step of introducing amino acid modifications related to interface control using charge repulsion or the like.

본 발명에 있어서의 「CH3영역의 안정성이 불안정」하다는 것은, Fc영역의 아미노산 잔기에 적어도 하나 이상의 개변을 가한 폴리펩티드의 호모체가 미개변의 폴리펩티드의 호모체에 비해 각 폴리펩티드 단체(單體)로 분리되기 쉬워져 있는 상태를 말한다. In the present invention, "the stability of the CH3 region is unstable" means that a homomorph of a polypeptide in which at least one modification is added to an amino acid residue in the Fc region is separated into individual polypeptides compared to a homomorph of an unmodified polypeptide. state that it is easy.

본 발명에 있어서의 「CH3영역의 안정성이 불안정」하다는 것은, 바람직하게는 pH 7.4에 있어서의 중쇄 CH3영역의 열변성 중간온도(Tm)가 72.5℃ 이하, 72.0℃ 이하, 71.5℃ 이하, 71.0℃ 이하, 70.5℃ 이하가 되도록, CH3영역의 아미노산 잔기에 개변을 도입한 상태를 말하고, 더욱 바람직하게는 70.4℃ 이하, 70.3℃ 이하, 70.2℃ 이하, 70.1℃ 이하, 70.0℃ 이하, 69.9℃ 이하, 69.8℃ 이하, 69.7℃ 이하, 69.6℃ 이하, 69.5℃ 이하, 69.0℃ 이하, 68.5℃ 이하, 68.0℃ 이하, 67.5℃ 이하가 되도록 CH3영역의 아미노산 잔기에 개변을 도입한 상태를 말한다. "The stability of the CH3 region is unstable" in the present invention means that the intermediate temperature (Tm) of thermal denaturation of the medium chain CH3 region at pH 7.4 is preferably 72.5°C or less, 72.0°C or less, 71.5°C or less, 71.0°C or less. Hereinafter, it refers to a state in which modifications are introduced into the amino acid residues of the CH3 region so as to be 70.5 ° C or less, more preferably 70.4 ° C or less, 70.3 ° C or less, 70.2 ° C or less, 70.1 ° C or less, 70.0 ° C or less, 69.9 ° C or less, 69.8 ° C or less, 69.7 ° C or less, 69.6 ° C or less, 69.5 ° C or less, 69.0 ° C or less, 68.5 ° C or less, 68.0 ° C or less, 67.5 ° C or less It refers to a state in which modifications are introduced into the amino acid residues of the CH3 region.

예를 들면 중쇄 CH3영역의 Tm은 본 명세서의 참고예 3에 기재된 방법으로 측정할 수 있고, 측정에 사용하는 완충액 등은 적절히 선택할 수 있다. For example, the Tm of the heavy chain CH3 region can be measured by the method described in Reference Example 3 of the present specification, and a buffer or the like used for the measurement can be appropriately selected.

본 발명의 폴리펩티드 간의 해리 및/또는 회합 제어방법의 더욱 바람직한 태양에 있어서는, 당해 방법은 중쇄 CH3영역의 EU 넘버링 397번 위치 및/또는 392번 위치에 아미노산 잔기의 변이를 도입하는 것을 특징으로 하는 방법이다. 당해 방법은 임의이고, 상기 전하 반발 등을 이용한 계면 제어에 관한 아미노산 개변을 도입하는 공정을 추가로 포함해도 된다. In a more preferred embodiment of the method for controlling dissociation and/or association between polypeptides of the present invention, the method is characterized by introducing a mutation of an amino acid residue at EU numbering position 397 and/or position 392 of the heavy chain CH3 region. to be. The method is optional, and may further include a step of introducing an amino acid modification for interface control using charge repulsion or the like.

본 발명의 비한정의 일태양으로서 마우스 유래의 폴리펩티드 간의 해리 및/또는 회합 제어방법에 있어서도 중쇄 CH3영역의 EU 넘버링 397번 위치 및/또는 392번 위치에 아미노산 잔기의 변이를 도입할 수 있다. 당해 방법은 임의이고, 상기 전하 반발 등을 이용한 계면 제어에 관한 아미노산 개변을 도입하는 공정을 추가로 포함해도 된다. As a non-limiting aspect of the present invention, amino acid residue mutations can be introduced at EU numbering position 397 and/or position 392 of the heavy chain CH3 region in a method for controlling dissociation and/or association between mouse-derived polypeptides. The method is optional, and may further include a step of introducing an amino acid modification for interface control using charge repulsion or the like.

397번 위치에 변이를 도입하는 아미노산 잔기로서는, 측쇄가 부피가 큰 아미노산 또는 측쇄 분지형 아미노산으로 개변하는 것이 바람직하다. As the amino acid residue for introducing a mutation at position 397, it is preferable to change the side chain to a bulky amino acid or branched side chain amino acid.

392번 위치에 변이를 도입하는 아미노산 잔기로서는, 음전하를 갖는 아미노산, 측쇄가 부피가 큰 아미노산 또는 측쇄 분지형 아미노산으로 개변하는 것이 바람직하다. As the amino acid residue for introducing a mutation at position 392, it is preferable to modify it with an amino acid having a negative charge, an amino acid having a bulky side chain, or a branched side chain amino acid.

본 발명에 있어서의 「측쇄가 부피가 큰 아미노산」으로서 Met(M), Phe(F)，Tyr(Y)，Val(V)，Leu(L)，Ile(I)，Trp(W), Arg(R), His(H), Glu(E), Lys(K), Gln(Q), Asp(D), Asn(N), Cys(C), Thr(T) 등을 들 수 있고, 바람직하게는 Met(M), Phe(F), Thr(T) 및 Tyr(Y)을 들 수 있다. Met (M), Phe (F), Tyr (Y), Val (V), Leu (L), Ile (I), Trp (W), Arg as "a bulky amino acid side chain" in the present invention (R), His(H), Glu(E), Lys(K), Gln(Q), Asp(D), Asn(N), Cys(C), Thr(T), etc. are mentioned, preferably Examples include Met(M), Phe(F), Thr(T) and Tyr(Y).

본 발명에 있어서의 「측쇄 분지형 아미노산」으로서 Val(V)，Ile(I)，Leu(L)를 들 수 있고, 바람직하게는 Val(V) 및 Ile(I) 등을 들 수 있다. Val(V), Ile(I), and Leu(L) are mentioned as "a branched chain amino acid" in this invention, Preferably Val(V), Ile(I), etc. are mentioned.

본 발명에 있어서의 「음전하를 갖는 아미노산」으로서 Asp(D) 및 Glu(E)를 들 수 있다. Asp (D) and Glu (E) are mentioned as "amino acid having a negative charge" in the present invention.

본 발명에 있어서의 「이종 다량체」로서 바람직하게는 다중특이성 항체 및 헤테로 융합 단백질 등을 들 수 있다. Preferred examples of the "heteromultimer" in the present invention include multispecific antibodies and heterofusion proteins.

본 발명의 비한정의 일태양으로서는，FcγR로의 결합이 증강되는 이종 다량체의 아미노산 개변을 제공하는 것이다. 바람직한 아미노산 개변 부위로서는, EU 넘버링 397번 위치의 아미노산의 개변을 들 수 있는데 이것에 한정되지는 않는다. 397번 위치에 변이를 도입하는 아미노산 잔기로서는, 측쇄가 부피가 큰 아미노산 또는 측쇄 분지형 아미노산으로 개변하는 것이 바람직하다. As a non-limiting aspect of the present invention, it is to provide an amino acid modification of a heteromultimer with enhanced binding to FcγR. Preferred amino acid modification sites include, but are not limited to, modifications of the amino acid at EU numbering position 397. As the amino acid residue for introducing a mutation at position 397, it is preferable to change the side chain to a bulky amino acid or branched side chain amino acid.

본 발명에 있어서 더욱 바람직하게는 다중특이성 항체로서 IgG type, scFv-IgG, Tandem scFv-Fc, DVD-Ig, Diabody-Fc, Single chain Diabody-Fc, IgG-scFv, sVD-IgG, Tandemab, scFv light chain C-terminal fusion, Tri-specific C-terminal fusion, Tri-specific N-terminal fusion, IgG-Fab 등을 들 수 있다(Bispecific Antibodies, Roland E. Kontermann, 2011, WO2010034441, WO2010145792). In the present invention, more preferably, as a multispecific antibody, IgG type, scFv-IgG, Tandem scFv-Fc, DVD-Ig, Diabody-Fc, Single chain Diabody-Fc, IgG-scFv, sVD-IgG, Tandemab, scFv light chain C-terminal fusion, Tri-specific C-terminal fusion, Tri-specific N-terminal fusion, IgG-Fab, and the like (Bispecific Antibodies, Roland E. Kontermann, 2011, WO2010034441, WO2010145792).

본 발명에 있어서 「항체」라는 용어는 가장 넓은 의미로 사용되고, 목적하는 생물학적 활성을 나타내는 한 단일클론 항체, 다중클론 항체, 항체 변이체(키메라 항체, 인간화 항체, 저분자화 항체(항체 단편도 포함한다), 다중특이성 항체 등)가 포함된다. 또한 본 발명에 있어서 「항체」는, 폴리펩티드 또는 이종 다량체 중 어느 것이어도 된다. 바람직한 항체는 단일클론 항체, 키메라 항체, 인간화 항체 및 항체 단편 등의 저분자화 항체이다. 본 발명에 있어서는 이들 항체의 취득 (제작) 시에 적합하게 본 발명의 해리 및/또는 회합 제어방법을 사용할 수 있다. In the present invention, the term "antibody" is used in the broadest sense, and monoclonal antibodies, polyclonal antibodies, and antibody variants (chimeric antibodies, humanized antibodies, low molecular weight antibodies (including antibody fragments)) , multispecific antibodies, etc.). In the present invention, the "antibody" may be either a polypeptide or a heteromultimer. Preferred antibodies are low molecular weight antibodies such as monoclonal antibodies, chimeric antibodies, humanized antibodies and antibody fragments. In the present invention, the dissociation and/or association control method of the present invention can be suitably used for the acquisition (production) of these antibodies.

본 발명의 방법에 제공되는 바람직한 폴리펩티드 또는 이종 다량체로서는, 예를 들면 항체의 중쇄 가변영역 및 경쇄 가변영역을 갖는 폴리펩티드 또는 이종 다량체를 들 수 있다. 또한 보다 바람직하게는 본 발명의 바람직한 태양으로서, 본 발명의 폴리펩티드 또는 이종 다량체가 2종 이상의 중쇄 가변영역과 2종 이상의 경쇄 가변영역을 포함할 때의 당해 폴리펩티드 또는 이종 다량체의 해리 및/또는 회합 제어방법을 들 수 있다. Preferred polypeptides or heteromultimers provided in the method of the present invention include, for example, polypeptides or heteromultimers having a heavy chain variable region and a light chain variable region of an antibody. Also more preferably, as a preferred embodiment of the present invention, when the polypeptide or heteromultimer of the present invention comprises two or more heavy chain variable regions and two or more light chain variable regions, dissociation and/or association of the polypeptide or heteromultimer control method.

본 발명의 항원 결합 활성을 갖는 폴리펩티드는 항체 중쇄의 아미노산 서열 또는 항체 경쇄의 아미노산 서열을 포함할 수 있다. 보다 구체적으로는, 제1 항원 결합 활성을 갖는 폴리펩티드 및 제2 항원 결합 활성을 갖는 폴리펩티드는 항체 중쇄의 아미노산 서열을 포함할 수 있고, 제3 항원 결합 활성을 갖는 폴리펩티드 및 제4 항원 결합 활성을 갖는 폴리펩티드는 항체 경쇄의 아미노산 서열을 포함할 수 있다. The polypeptide having antigen-binding activity of the present invention may include an amino acid sequence of an antibody heavy chain or an amino acid sequence of an antibody light chain. More specifically, a polypeptide having a first antigen-binding activity and a polypeptide having a second antigen-binding activity may include an amino acid sequence of an antibody heavy chain, and a polypeptide having a third antigen-binding activity and a polypeptide having a fourth antigen-binding activity The polypeptide may comprise the amino acid sequence of an antibody light chain.

또한 목적의 폴리펩티드 다량체가 제1 폴리펩티드와 제3 폴리펩티드로 이량체를 형성하고, 제2 폴리펩티드와 제4 폴리펩티드로 이량체를 형성하며, 추가로 이들 이량체끼리가 다량체를 형성하는 4량체인 경우에는, 본 발명의 폴리펩티드 다량체로서, 예를 들면 제1과 제2 항원 결합 활성을 갖는 폴리펩티드가 항체 중쇄의 아미노산 서열을 포함하는 폴리펩티드이고, 제3과 제4 항원 결합 활성을 갖는 폴리펩티드가 항체 경쇄의 아미노산 서열을 포함하는 폴리펩티드인 폴리펩티드 다량체를 사용하는 것도 가능하다. Further, when the target polypeptide multimer is a tetramer in which a first polypeptide and a third polypeptide form a dimer, a second polypeptide and a fourth polypeptide form a dimer, and these dimers form a multimer For example, as a polypeptide multimer of the present invention, a polypeptide having first and second antigen-binding activity is a polypeptide comprising an amino acid sequence of an antibody heavy chain, and a polypeptide having a third and fourth antigen-binding activity is an antibody light chain It is also possible to use a polypeptide multimer, which is a polypeptide comprising the amino acid sequence of

본 발명에 있어서 더욱 바람직하게는 다중특이성 항체로서 이중특이성 항체를 들 수 있다. In the present invention, more preferably, as a multispecific antibody, a bispecific antibody is mentioned.

즉, 본 발명의 바람직한 태양에 있어서는, 본 발명은 예를 들면 2종의 중쇄(본 발명에 있어서의 폴리펩티드 다량체의 제1 폴리펩티드와 제2 폴리펩티드)와, 2종의 경쇄(본 발명에 있어서의 폴리펩티드 다량체의 제3 폴리펩티드와 제4 폴리펩티드)로 구성되는 이중특이성 항체에 대해서 해리 및/또는 회합을 제어하는 방법에 관한 것이다. That is, in a preferred embodiment of the present invention, the present invention provides, for example, two types of heavy chains (the first polypeptide and the second polypeptide of the polypeptide multimer in the present invention) and two types of light chains (in the present invention) A method of controlling dissociation and/or association for a bispecific antibody consisting of a third polypeptide and a fourth polypeptide) of a polypeptide multimer.

본 발명의 바람직한 태양의 「이중특이성 항체」에 대해서 추가로 상세하게 설명하면, 상기 「제1 폴리펩티드 및 제2 폴리펩티드」란, 항체를 형성하는 2개의 중쇄(H쇄) 중 한쪽 H쇄(제1 H쇄) 및 당해 H쇄와는 상이한 다른 한쪽 H쇄(제2 H쇄)를 말한다. 즉, 2개의 H쇄 중 임의로 어느 한쪽을 제1 H쇄로 하고, 다른 쪽을 제2 H쇄로 할 수 있다. 마찬가지로 「제3 폴리펩티드 및 제4 폴리펩티드」란, 이중특이성 항체를 형성하는 2개의 경쇄(L쇄) 중 한쪽 L쇄(제1 L쇄) 및 당해 L쇄와는 상이한 다른 한쪽 L쇄(제2 L쇄)를 가리키며, 2개의 L쇄 중 어느 한쪽을 임의로 제1 L쇄로 하고, 다른 쪽을 제2 L쇄로 할 수 있다. 통상 제1 L쇄와 제1 H쇄는 어떤 항원(또는 에피토프)을 인식하는 동일 항체로부터 유래하고, 제2 L쇄와 제2 H쇄도 어떤 항원(또는 에피토프)을 인식하는 동일 항체로부터 유래한다. 여기서, 제1 H쇄·L쇄로 형성되는 L쇄-H쇄쌍을 제1 쌍(또는 제1 HL 분자), 제2 H쇄·L쇄로 형성되는 L쇄-H쇄쌍을 제2 쌍(또는 제2 HL 분자)이라 부른다. 제1 쌍과 제2 쌍이 인식하는 항원은 동일해도 되는데, 바람직하게는 다른 에피토프를 인식한다. 이 경우, 제1 쌍 및 제2 쌍의 H쇄와 L쇄는 서로 다른 아미노산 서열을 가지고 있는 것이 바람직하다. 제1 쌍과 제2 쌍이 다른 에피토프를 인식하는 경우, 그 제1 쌍과 제2 쌍은 전혀 다른 항원을 인식해도 되고, 동일 항원 상의 다른 부위(다른 에피토프)를 인식해도 된다(예를 들면 항원이 헤테로 수용체인 경우에는 다중특이성 항체는 헤테로 수용체를 구성하는 다른 도메인을 인식하거나, 또는 항원이 모노머인 경우에는 다중특이성 항체는 모노머 항원의 복수 개소를 인식한다). 통상, 이러한 분자는 2개의 항원과 결합하는 것인데, 그 이상의 (예를 들면 3종류의) 항원에 대해 특이성을 가지고 있어도 된다. 또한 한쪽이 단백질, 펩티드, 유전자, 당 등의 항원을 인식하고, 다른 쪽이 방사성 물질, 화학요법제, 세포 유래 톡신 등의 세포상해성 물질 등을 인식해도 된다. 그러나 특정 H쇄와 L쇄의 조합으로 형성되는 쌍을 갖는 항체를 제작하고자 생각한 경우에는, 그 특정 H쇄와 L쇄를 제1 쌍 및 제2 쌍으로서 임의로 결정할 수 있다. The "bispecific antibody" of the preferred embodiment of the present invention will be described in further detail. The "first polypeptide and second polypeptide" means one of the two heavy chains (H chains) forming the antibody (the first H chain) H chain) and the other H chain (second H chain) different from the H chain. That is, one of the two H chains may be arbitrarily used as the first H chain, and the other may be used as the second H chain. Similarly, "third polypeptide and fourth polypeptide" means one L chain (first L chain) of the two light chains (L chains) forming the bispecific antibody and the other L chain (second L chain) different from the L chain chain), and either one of the two L chains can be arbitrarily used as the first L chain, and the other can be used as the second L chain. Usually, the first L chain and the first H chain are derived from the same antibody recognizing an antigen (or epitope), and the second L chain and the second H chain are also derived from the same antibody recognizing a certain antigen (or epitope). Here, the L chain-H chain pair formed from the first H chain/L chain is the first pair (or the first HL molecule), and the L chain-H chain pair formed from the second H chain/L chain is the second pair (or the second pair) HL molecules). The antigens recognized by the first pair and the second pair may be the same, but preferably recognize different epitopes. In this case, it is preferable that the H chain and the L chain of the first pair and the second pair have different amino acid sequences. When the first pair and the second pair recognize different epitopes, the first pair and the second pair may recognize completely different antigens or may recognize different sites (different epitopes) on the same antigen (for example, antigens In the case of a heteroreceptor, the multispecific antibody recognizes other domains constituting the heteroreceptor, or in the case where the antigen is a monomer, the multispecific antibody recognizes multiple sites of the monomeric antigen). Usually, such a molecule binds to two antigens, but may have specificity for more (for example, three types) antigens. Moreover, one side may recognize antigens, such as a protein, a peptide, a gene, and a sugar, and the other side may recognize a cytotoxic substance, such as a radioactive substance, a chemotherapeutic agent, a cell-derived toxin, etc. However, when it is intended to produce an antibody having a pair formed by a combination of a specific H chain and L chain, the specific H chain and L chain can be arbitrarily determined as the first pair and the second pair.

본 발명에 있어서의 「융합 단백질」은 2개 이상의 동일 또는 실질적으로 유사한 단백질 분자가 Ig 힌지영역 아미노산 서열 링커를 매개로 접합된 단백질을 설명한다. 1종을 초과하는 단백질을 함유한 융합 단백질을 설명하는 경우, 「헤테로」의 접두어를 사용한다. 예를 들면 「헤테로 융합 단백질」은 1 이상의 잔부 단백질과 다른 1 이상의 단백질을 함께 접합한 2개 이상의 단백질을 함유한다. The "fusion protein" in the present invention describes a protein in which two or more identical or substantially similar protein molecules are joined via an Ig hinge region amino acid sequence linker. When describing a fusion protein containing more than one protein, the prefix "hetero" is used. For example, a "hetero fusion protein" contains two or more proteins in which one or more residual proteins and one or more other proteins are joined together.

본 발명에 있어서의 「항체」에는 전술한 항체에 대해 추가로 아미노산의 치환, 결실, 부가 및/또는 삽입 또는 키메라화나 인간화 등에 의해 그 아미노산 서열이 개변된 것이 포함된다. 아미노산의 치환, 결실, 부가 및/또는 삽입 및 인간화, 키메라화 등의 아미노산 서열의 개변은 당업자에게 공지의 방법으로 행하는 것이 가능하다. 마찬가지로 본 발명에 있어서의 항체를 재조합 항체로서 제작할 때 이용하는 항체의 가변영역 및 불변영역도 아미노산의 치환, 결실, 부가 및/또는 삽입 또는 키메라화나 인간화 등에 의해 그 아미노산 서열을 개변해도 된다. The "antibody" in the present invention includes those whose amino acid sequence has been altered by substitution, deletion, addition and/or insertion of amino acids, chimerization, humanization, or the like of the above-mentioned antibody. Amino acid substitution, deletion, addition and/or insertion, and alteration of the amino acid sequence such as humanization and chimerization can be performed by methods known to those skilled in the art. Similarly, the variable region and constant region of an antibody used when preparing the antibody of the present invention as a recombinant antibody may also be modified in amino acid sequence by amino acid substitution, deletion, addition and/or insertion, chimerization, humanization, or the like.

본 발명에 있어서의 항체는 마우스 항체, 인간 항체, 랫트 항체, 토끼 항체, 염소 항체, 낙타 항체 등, 어떠한 동물 유래의 항체여도 된다. 또한 예를 들면 키메라 항체, 그 중에서도 인간화 항체 등의 아미노산 서열을 치환한 개변 항체여도 된다. 또한 각종 분자를 결합시킨 항체 수식물, 항체 단편, 저분자화 항체 등 어떠한 항체여도 된다. The antibody in the present invention may be an antibody derived from any animal, such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, and a camel antibody. Alternatively, for example, a chimeric antibody, particularly a modified antibody obtained by substituting an amino acid sequence such as a humanized antibody may be used. Also, any antibody such as modified antibody, antibody fragment, and low-molecular-weight antibody to which various molecules are bound may be used.

「키메라 항체」란 다른 동물 유래의 서열을 조합하여 제작되는 항체이다. 예를 들면 마우스 항체의 중쇄, 경쇄의 가변(V)영역과 인간 항체의 중쇄, 경쇄의 불변(C)영역으로 이루어지는 항체를 예시할 수 있다. 키메라 항체의 제작은 공지로, 예를 들면 항체 V영역을 코드하는 DNA를 인간 항체 C영역을 코드하는 DNA와 연결하고, 이를 발현 벡터에 삽입하고 숙주에 도입하여 생산시킴으로써 키메라 항체를 얻을 수 있다. A "chimeric antibody" is an antibody produced by combining sequences derived from different animals. For example, an antibody comprising the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody can be exemplified. The production of a chimeric antibody is known, for example, by ligating DNA encoding the antibody V region with DNA encoding the human antibody C region, inserting it into an expression vector, introducing it into a host, and producing a chimeric antibody.

「인간화 항체」란 재구성(reshaped) 인간 항체로도 칭해지는, 인간 이외의 포유동물 유래의 항체, 예를 들면 마우스 항체의 상보성 결정영역(CDR;complementarity determining region)을 인간 항체의 CDR로 이식한 것이다. CDR을 동정하기 위한 방법은 공지이다(Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877). 또한 그 일반적인 유전자 재조합 수법도 공지이다(유럽 특허출원 공개번호 EP 125023호 공보, WO 96/02576호 공보 참조). 이에 공지의 방법으로, 예를 들면 마우스 항체의 CDR을 결정하고, 그 CDR과 인간 항체의 프레임워크 영역(framework region；FR)이 연결된 항체를 코드하는 DNA를 취득하여, 인간화 항체를 통상의 발현 벡터를 사용한 시스템에 의해 생산할 수 있다. 이러한 DNA는 CDR 및 FR 양쪽의 말단영역에 오버랩되는 부분을 갖도록 제작한 여러 개의 올리고 뉴클레오티드를 프라이머로서 사용하여 PCR법에 의해 합성할 수 있다(WO98/13388호 공보에 기재된 방법을 참조). CDR을 매개로 연결되는 인간 항체의 FR은 CDR이 양호한 항원 결합 부위를 형성하도록 선택된다. 필요에 따라 재구성 인간 항체의 CDR이 적절한 항원 결합 부위를 형성하도록, 항체의 가변영역에 있어서의 FR의 아미노산을 개변해도 된다(Sato et al., Cancer Res. (1993) 53: 851-6). 개변할 수 있는 FR 중의 아미노산 잔기에는 항원에 직접, 비공유 결합에 의해 결합하는 부분(Amit et al., Science (1986) 233: 747-53), CDR 구조에 영향 또는 작용하는 부위(Chothia et al., J. Mol. Biol. (1987) 196: 901-17) 및 VH-VL 상호작용에 관련된 부분(EP239400호 특허공보)이 포함된다. A "humanized antibody" is a non-human mammal-derived antibody, also referred to as a reshaped human antibody, in which the complementarity determining region (CDR) of a mouse antibody is grafted into the CDR of a human antibody. . Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342:877). In addition, the general genetic recombination method is also known (see European Patent Application Publication No. EP 125023, WO 96/02576). Thus, by a known method, for example, the CDRs of a mouse antibody are determined, DNA encoding the antibody in which the CDRs and the framework regions of the human antibody are linked are obtained, and the humanized antibody is converted into a conventional expression vector. can be produced by a system using Such DNA can be synthesized by PCR using several oligonucleotides prepared to have overlapping portions at the end regions of both CDR and FR as primers (refer to the method described in WO98/13388). The FRs of human antibodies that are linked via CDRs are selected such that the CDRs form a favorable antigen binding site. If necessary, the amino acid of the FR in the variable region of the antibody may be altered so that the CDRs of the reconstituted human antibody form an appropriate antigen-binding site (Sato et al., Cancer Res. (1993) 53: 851-6). Amino acid residues in the FR that can be modified include a portion directly or non-covalently binding to an antigen (Amit et al., Science (1986) 233: 747-53), a portion that affects or acts on the CDR structure (Chothia et al. , J. Mol. Biol. (1987) 196: 901-17) and parts related to VH-VL interactions (EP239400 Patent Publication).

본 발명에 있어서의 항체가 키메라 항체 또는 인간화 항체인 경우에는, 이들 항체의 C영역에는 바람직하게는 인간 항체 유래의 것이 사용된다. 예를 들면 H쇄의 경우는, Cγ1, Cγ2, Cγ3, Cγ4를, L쇄의 경우는 Cκ, Cλ를 사용할 수 있다. 또한 항체 또는 그의 생산의 안정성을 개선하기 위해 인간 항체 C영역을 필요에 따라 수식해도 된다. 본 발명에 있어서의 키메라 항체는 바람직하게는 인간 이외의 포유동물 유래 항체의 가변영역과 인간 항체 유래의 불변영역으로 이루어진다. 한편, 인간화 항체는 바람직하게는 인간 이외의 포유동물 유래 항체의 CDR과, 인간 항체 유래의 FR 및 C영역으로 이루어진다. 인간 항체 유래의 불변영역은 IgG(IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD 및 IgE 등의 아이소타입마다 고유의 아미노산 서열을 갖는다. 본 발명에 있어서의 인간화 항체에 사용되는 불변영역은 어느 아이소타입에 속하는 항체의 불변영역이어도 된다. 바람직하게는 인간 IgG의 불변영역이 사용되는데 이에 한정되는 것은 아니다. 또한 인간화 항체에 이용되는 인간 항체 유래의 FR도 특별히 한정되지 않고, 어느 아이소타입에 속하는 항체의 것이어도 된다. When the antibody in the present invention is a chimeric antibody or a humanized antibody, those derived from a human antibody are preferably used for the C region of these antibodies. For example, in the case of the H chain, Cγ1, Cγ2, Cγ3, Cγ4, and in the case of the L chain, Cκ and Cλ can be used. Further, in order to improve the stability of the antibody or its production, the human antibody C region may be modified as necessary. The chimeric antibody in the present invention is preferably composed of a variable region derived from a non-human mammal and a constant region derived from a human antibody. On the other hand, the humanized antibody preferably consists of CDRs of an antibody derived from a mammal other than human and FR and C regions derived from a human antibody. The human antibody-derived constant region has a unique amino acid sequence for each isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD and IgE. The constant region used for the humanized antibody in the present invention may be an antibody constant region belonging to any isotype. Preferably, the constant region of human IgG is used, but is not limited thereto. Moreover, the FR derived from a human antibody used for a humanized antibody is not specifically limited, either, The thing of the antibody belonging to any isotype may be sufficient.

본 발명에 있어서의 키메라 항체 및 인간화 항체의 가변영역 및 불변영역은 원래의 항체의 결합 특이성을 나타내는 한 결실, 치환, 삽입 및/또는 부가 등에 의해 개변되어 있어도 된다. The variable regions and constant regions of the chimeric antibody and humanized antibody in the present invention may be modified by deletion, substitution, insertion, and/or addition, etc. as long as they show the binding specificity of the original antibody.

인간 유래의 서열을 이용한 키메라 항체 및 인간화 항체는 인간 체내에 있어서의 항원성이 저하되어 있기 때문에, 치료 목적 등으로 인간에 투여하는 경우에 유용할 것으로 생각된다. Chimeric antibodies and humanized antibodies using human-derived sequences are thought to be useful when administered to humans for therapeutic purposes or the like because their antigenicity in the human body is reduced.

등전점 개변 기술 등과의 조합Combination with isoelectric point change technology, etc.

본 발명의 추가적인 바람직한 태양으로서는 폴리펩티드의 등전점(pI값)을 개변하는 아미노산 변이를 본 발명의 폴리펩티드에 도입함으로써, 목적의 제1~제4 폴리펩티드를 갖는 폴리펩티드 다량체를 보다 고순도로 또한 효율적으로 정제 또는 제조하는 것이 가능하다(WO2007114325, US20130171095). 폴리펩티드의 회합을 촉진하기 위해 도입되는 아미노산 변이로서는 Protein Eng. 1996 Jul;9(7):617-21., Protein Eng Des Sel. 2010 Apr;23(4):195-202., J Biol Chem. 2010 Jun 18;285(25):19637-46., WO2009080254, US20130195849 등에 기재된 중쇄 불변영역의 CH3 도메인의 개변에 의해 2종류의 중쇄 불변영역을 포함하는 폴리펩티드를 헤테로 회합화하는 방법, 및 WO2009080251, WO2009080252, WO2009080253 등에 기재된 중쇄와 경쇄의 특정 조합의 회합화를 촉진하는 방법 등을 사용하는 것도 가능하다. In a further preferred embodiment of the present invention, by introducing into the polypeptide of the present invention an amino acid mutation that alters the isoelectric point (pI value) of the polypeptide, a polypeptide multimer having the first to fourth polypeptides of interest can be purified or efficiently purified or It is possible to prepare (WO2007114325, US20130171095). As amino acid mutations introduced to promote the association of polypeptides, Protein Eng. 1996 Jul;9(7):617-21., Protein Eng Des Sel. 2010 Apr;23(4):195-202., J Biol Chem. 2010 Jun 18;285(25):19637-46., WO2009080254, a method of hetero-associating a polypeptide comprising two types of heavy chain constant regions by modification of the CH3 domain of the heavy chain constant region described in WO2009080254, US20130195849, etc., and WO2009080251, WO2009080252 , a method for promoting association of a specific combination of a heavy chain and a light chain described in WO2009080253 and the like can also be used.

표적 조직 특이적 항원 결합 분자에 관한 기술과의 조합Combination with technology for target tissue specific antigen binding molecules

본 발명의 비한정의 실시태양으로서는, 표적 조직 특이적으로 존재하는 분자의 농도 의존적으로 항원으로부터 해리·결합하는 항체 기술(WO2013/180200)과의 조합을 들 수 있다. As a non-limiting embodiment of the present invention, a combination with an antibody technology (WO2013/180200) that dissociates and binds from an antigen in a concentration-dependent manner of a target tissue-specific molecule (WO2013/180200) is exemplified.

다른 불변영역 및/또는 가변영역 개변 기술과의 조합Combination with other constant region and / or variable region modification technology

본 발명의 비한정의 실시태양으로서는, FcγR로의 결합 증강을 목적으로 한 불변영역 개변 기술(WO2013047752)과의 조합을 들 수 있다. As a non-limiting embodiment of the present invention, a combination with constant region modification technology (WO2013047752) for the purpose of enhancing binding to FcγR is mentioned.

본 발명과 다른 불변영역 개변 기술의 기타 조합의 태양으로서 보체로의 결합을 제어하는 기술과의 조합을 들 수 있다. 보체로서는 보체 캐스케이드를 형성하는 폴리펩티드라면 어느 보체 성분도 사용될 수 있는데, 바람직한 보체로서는 옵소닌의 결합에 관여하는 C1q, C1r 또는 C1s의 보체 성분을 적합하게 들 수 있다. Fc영역의 보체에 대한 결합 활성이 보체에 대한 천연형 Fc영역의 결합 활성보다도 높은 Fc영역은 천연형 Fc영역의 아미노산을 개변함으로써 제작될 수 있다. 여기서 말하는 천연형 Fc영역이란, 인간 IgG1, IgG2, IgG3 또는 IgG4로 표시되는 Fc영역을 가리킨다. Fc영역의 보체에 대한 결합 활성이 천연형 Fc영역의 보체에 대한 결합 활성보다 높은지 여부는 FACS, ELISA 등의 공지의 면역학적 방법을 사용하여 적절히 실시될 수 있다. Fc영역의 「아미노산의 개변」 또는 「아미노산 개변」이란, 출발 Fc영역의 아미노산 서열과는 다른 아미노산 서열로 개변하는 것을 포함한다. 출발 Fc영역의 수식 개변체가 pH 중성역에 있어서 보체에 결합할 수 있는 한, 어느 Fc영역도 출발 도메인으로서 사용될 수 있다. 또한 이미 개변이 가해진 Fc영역을 출발 Fc영역으로 하여 추가적인 개변이 가해진 Fc영역도 본 발명의 Fc영역으로서 적합하게 사용될 수 있다. 출발 Fc영역이란, 폴리펩티드 그 자체, 출발 Fc영역을 포함하는 조성물, 또는 출발 Fc영역을 코드하는 아미노산 서열을 의미할 수 있다. 출발 Fc영역에는 항체의 항목에서 개설된 재조합에 의해 생산된 공지의 IgG 항체의 Fc영역이 포함될 수 있다. 출발 Fc영역의 기원은 한정되지 않지만 비인간 동물의 임의의 생물 또는 인간으로부터 취득될 수 있다. 바람직하게는 임의의 생물로서는 마우스, 랫트, 모르모트, 햄스터, 게르빌루스쥐, 고양이, 토끼, 개, 염소, 양, 소, 말, 낙타 및 비인간 영장류로부터 선택되는 생물을 적합하게 들 수 있다. 다른 태양에 있어서, 출발 Fc영역은 또한 게잡이원숭이, 마모셋, 빨간털원숭이, 침팬지 또는 인간으로부터 취득될 수 있다. 바람직하게는 출발 Fc영역은 인간 IgG1으로부터 취득될 수 있는데, IgG의 특정 클래스에 한정되는 것도 아니다. 이는 인간 IgG1, IgG2, IgG3 또는 IgG4의 Fc영역을 출발 Fc영역으로서 적절히 사용할 수 있는 것을 의미한다. 마찬가지로 본 명세서에 있어서 상기 임의의 생물로부터의 IgG의 임의의 클래스 또는 서브클래스의 Fc영역을 바람직하게는 출발 Fc영역으로서 사용할 수 있는 것을 의미한다. 천연에 존재하는 IgG의 배리언트 또는 조작된 형의 예는, 공지의 문헌(Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, WO2009086320, WO2008092117, WO2007041635 및 WO2006105338)에 기재되어 있는데 이들에 한정되지 않는다. As an aspect of other combinations of the present invention and other constant region modification technology, a combination with a technology for controlling binding to complement is mentioned. Any complement component can be used as the complement as long as it is a polypeptide that forms the complement cascade. Preferred complements include C1q, C1r or C1s complement components involved in opsonin binding. An Fc region in which the binding activity of the Fc region to complement is higher than that of the native Fc region to complement can be produced by modifying the amino acids of the native Fc region. The native Fc region as used herein refers to an Fc region represented by human IgG1, IgG2, IgG3 or IgG4. Whether the complement-binding activity of the Fc region is higher than the complement-binding activity of the native Fc region can be appropriately carried out using known immunological methods such as FACS and ELISA. "Modification of amino acid" or "modification of amino acid" of the Fc region includes alteration with an amino acid sequence different from the amino acid sequence of the starting Fc region. Any Fc region may be used as the starting domain as long as the modified variant of the starting Fc region can bind to complement in the neutral pH region. In addition, the Fc region to which the already modified Fc region has been added as the starting Fc region, additional modifications are applied can also be suitably used as the Fc region of the present invention. The starting Fc region may refer to a polypeptide itself, a composition including the starting Fc region, or an amino acid sequence encoding the starting Fc region. The starting Fc region may include the Fc region of a known IgG antibody produced by recombination outlined in the section of the antibody. The origin of the starting Fc region is not limited, but may be obtained from any organism of a non-human animal or a human. Preferable examples of the arbitrary organisms include organisms selected from mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, cattle, horses, camels and non-human primates. In another embodiment, the starting Fc region may also be obtained from a cynomolgus monkey, a marmoset, a rhesus monkey, a chimpanzee or a human. Preferably, the starting Fc region can be obtained from human IgG1, but is not limited to a specific class of IgG. This means that the Fc region of human IgG1, IgG2, IgG3 or IgG4 can be appropriately used as the starting Fc region. Similarly, in the present specification, it means that an Fc region of any class or subclass of IgG from any of the above organisms can be preferably used as a starting Fc region. Examples of naturally occurring variants or engineered forms of IgG are described in the known literature (Curr. Opin. Biotechnol. (2009) 20 (6), 685-91, Curr. Opin. Immunol. (2008) 20 (4) ), 460-470, Protein Eng. Des. Sel. (2010) 23 (4), 195-202, WO2009086320, WO2008092117, WO2007041635 and WO2006105338).

아미노산의 개변은 보체에 대한 결합 활성을 갖거나 또는 보체 결합에 대한 결합 활성을 높일 수 있는 한 어떠한 위치의 아미노산도 개변될 수 있다. 항원 결합 분자가 인간 Fc영역으로서 인간 IgG1의 Fc영역을 포함하고 있는 경우, 보체에 대한 결합이 인간 IgG1의 출발 Fc영역의 결합 활성보다 증강되는 효과를 초래하는 개변이 포함되어 있는 것이 바람직하다. 보체에 대한 결합 활성을 개변하기 위한 아미노산으로서는, 예를 들면 Duncan 등(Nature (1988) 332, 738-740), Tao 등(J. Exp. Med.(1993) 178, 661-667), Brekke 등(Eur. J. Immunol. (1994) 24, 2542-2547), Xu 등(Immunol. (1993) 150, 152A), WO1994029351, WO2000042072 및 WO2011091078 등에 있어서 보고되어 있는 C1q에 대한 결합 활성이 개변된 Fc영역의 아미노산이 예시된다. Amino acids can be modified at any position as long as the amino acid has a binding activity for complement or can increase the binding activity for complement binding. When the antigen-binding molecule contains an Fc region of human IgG1 as a human Fc region, it is preferable that a modification that results in an effect that enhances the binding activity of the human IgG1 starting Fc region in binding to complement is included. As amino acids for modifying the binding activity to complement, for example, Duncan et al. (Nature (1988) 332, 738-740), Tao et al. (J. Exp. Med. (1993) 178, 661-667), Brekke et al. (Eur. J. Immunol. (1994) 24, 2542-2547), Xu et al. (Immunol. (1993) 150, 152A), WO1994029351, WO2000042072 and WO2011091078 reported in such as C1q binding activity is modified Fc region of amino acids are exemplified.

그러한 C1q에 대한 결합 활성이 증강되는 개변이 가능한 아미노산으로서, 예를 들면 EU 넘버링으로 표시되는 231번 위치에서 238번 위치, 318번 위치에서 337번 위치로부터 선택되는 적어도 하나 이상의 아미노산을 들 수 있다. 당해 아미노산의 비한정의 일례로서 235번 위치, 237번 위치, 267번 위치, 268번 위치, 276번 위치, 318번 위치, 320번 위치, 322번 위치, 324번 위치, 327번 위치, 331번 위치 및 333번 위치의 군으로부터 선택되는 적어도 하나 이상의 아미노산을 들 수 있다. 이들 아미노산의 개변에 의해 IgG형 면역글로불린의 Fc영역의 보체에 대한 결합이 증강된다. As an amino acid that can be modified to enhance the binding activity to such C1q, for example, at least one or more amino acids selected from positions 231 to 238 and 318 to 337 as shown in EU numbering. As a non-limiting example of the amino acid, position 235, position 237, position 267, position 268, position 276, position 318, position 320, position 322, position 324, position 327, position 331 and at least one amino acid selected from the group of position and position 333. Binding to the complement of the Fc region of IgG-type immunoglobulin is enhanced by alteration of these amino acids.

특히 바람직한 개변으로서는, 예를 들면 Fc영역의 EU 넘버링으로 표시되는；As a particularly preferred modification, for example, represented by EU numbering of the Fc region;

267번 위치의 아미노산이 Glu, The amino acid at position 267 is Glu,

268번 위치의 아미노산이 Phe 또는 Tyr 중 어느 하나, the amino acid at position 268 is either Phe or Tyr,

276번 위치의 아미노산이 Arg, The amino acid at position 276 is Arg,

324번 위치의 아미노산이 Thr, The amino acid at position 324 is Thr,

327번 위치의 아미노산이 Gly, The amino acid at position 327 is Gly,

331번 위치의 아미노산이 Pro 또는 The amino acid at position 331 is Pro or

333번 위치의 아미노산이 Ala, Asp, Gly, Ser 또는 Val 중 어느 하나The amino acid at position 333 is any one of Ala, Asp, Gly, Ser or Val

를 들 수 있다. 또한 개변되는 아미노산의 수는 특별히 한정되지 않고, 1개소만의 아미노산이 개변될 수 있고, 이들이 임의로 조합된 2개소 이상의 아미노산이 개변될 수 있다. can be heard In addition, the number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, and two or more amino acids in which they are arbitrarily combined may be modified.

본 발명과 다른 불변영역 개변 기술의 기타 조합의 태양으로서, 산성 pH에서의 FcRn 결합 증강 개변 Fc기술(WO2002060919, WO2004035752, WO2000042072), 중성 pH에서의 FcRn 결합 증강 개변 Fc기술(WO2011122011, WO2012133782), 억제형 Fcγ 수용체 선택적 결합 증강 기술(WO2012115241, WO2013125667), 활성형 Fcγ 수용체 선택적 결합 증강 기술(ADCC 활성 증강 기술)(WO2013002362), 류머티즘 인자(Rheumatoid factor)로의 결합 활성을 저하시키는 기술(WO2013046704) 등의 항체 개변 기술과의 조합을 들 수 있다. As an aspect of other combinations of the present invention and other constant region modification technology, FcRn binding enhancement modified Fc technology at acidic pH (WO2002060919, WO2004035752, WO2000042072), FcRn binding enhancement modified Fc technology at neutral pH (WO2011122011, WO2012133782), inhibition Antibodies such as type Fcγ receptor selective binding enhancement technology (WO2012115241, WO2013125667), active Fcγ receptor selective binding enhancement technology (ADCC activity enhancement technology) (WO2013002362), rheumatoid factor (Rheumatoid factor), and lowering technology (WO2013046704) Combination with modification technology is mentioned.

본 발명과 가변영역 개변 기술의 비한정 조합의 태양으로서는, pH 의존성 항체(WO2009125825), 칼슘 의존성 항체(WO2012073992) 등의 개변 기술과의 조합을 들 수 있다. As an aspect of the non-limiting combination of this invention and a variable region modification technique, a combination with modification techniques, such as a pH-dependent antibody (WO2009125825) and a calcium-dependent antibody (WO2012073992), is mentioned.

항체 라이브러리, 면역 및 하이브리도마 제작Antibody library, immunization and hybridoma construction

본 발명의 방법에 있어서의 변이 도입 전의 항체(본 명세서에 있어서는, 단순히 「본 발명의 항체」로 기재하는 경우 있음)의 H쇄 또는 L쇄를 코드하는 유전자로서 기지의 서열을 사용하는 것도 가능하고, 또한 당업자에게 공지의 방법으로 취득하는 것도 가능하다. 예를 들면 항체 라이브러리로부터 취득하는 것도 가능하고, 단일클론 항체를 생산하는 하이브리도마로부터 항체를 코드하는 유전자를 클로닝하여 취득하는 것도 가능하다. It is also possible to use a known sequence as a gene encoding the H chain or L chain of the antibody (in the present specification, it is simply described as "antibody of the present invention") before the mutation is introduced in the method of the present invention. , it is also possible to obtain it by a method known to those skilled in the art. For example, it can be obtained from an antibody library, or it can be obtained by cloning a gene encoding an antibody from a hybridoma producing a monoclonal antibody.

항체 라이브러리에 대해서는 이미 많은 항체 라이브러리가 공지되어 있고, 또한 항체 라이브러리의 제작방법도 공지이기 때문에 당업자는 적절히 항체 라이브러리를 입수하는 것이 가능하다. 예를 들면 항체 파지 라이브러리에 대해서는 Clackson et al., Nature 1991, 352: 624-8, Marks et al., J. Mol. Biol. 1991, 222: 581-97, Waterhouses et al., Nucleic Acids Res. 1993, 21: 2265-6, Griffiths et al., EMBO J. 1994, 13: 3245-60, Vaughan et al., Nature Biotechnology 1996, 14: 309-14 및 일본국 특허공표 평20-504970호 공보 등의 문헌을 참조할 수 있다. 그 밖에, 진핵세포를 라이브러리로 하는 방법(WO95/15393호 팸플릿)이나 리보솜 제시법 등의 공지의 방법을 사용하는 것이 가능하다. 또한 인간 항체 라이브러리를 사용하여 패닝에 의해 인간 항체를 취득하는 기술이 알려져 있다. 예를 들면 인간 항체의 가변영역을 단일 사슬 항체(scFv)로서 파지 디스플레이법에 의해 파지의 표면에 발현시키고, 항원에 결합하는 파지를 선택할 수 있다. 선택된 파지의 유전자를 해석하면, 항원에 결합하는 인간 항체의 가변영역을 코드하는 DNA 서열을 결정할 수 있다. 항원에 결합하는 scFv의 DNA 서열이 명확해지면, 당해 서열을 토대로 적당한 발현 벡터를 제작하여 인간 항체를 취득할 수 있다. 이들 방법은 이미 주지로서 WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388을 참고로 할 수 있다. As for the antibody library, many antibody libraries are already known, and since the preparation method of an antibody library is also known, it is possible for those skilled in the art to obtain an antibody library suitably. For example, for antibody phage libraries, see Clackson et al., Nature 1991, 352: 624-8, Marks et al., J. Mol. Biol. 1991, 222: 581-97, Waterhouses et al., Nucleic Acids Res. 1993, 21: 2265-6, Griffiths et al., EMBO J. 1994, 13: 3245-60, Vaughan et al., Nature Biotechnology 1996, 14: 309-14 and Japanese Patent Publication No. Hei 20-504970, etc. Reference can be made to the literature of In addition, it is possible to use a known method such as a method using eukaryotic cells as a library (Pamphlet WO95/15393) or a ribosome presentation method. Also known is a technique for obtaining human antibodies by panning using a human antibody library. For example, the variable region of a human antibody can be expressed as a single-chain antibody (scFv) on the surface of a phage by a phage display method, and a phage that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of a human antibody that binds to an antigen can be determined. When the DNA sequence of the scFv that binds to the antigen is clarified, an appropriate expression vector can be prepared based on the sequence to obtain a human antibody. These methods are already well known and reference may be made to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388.

하이브리도마로부터 항체를 코드하는 유전자를 취득하는 방법은 기본적으로는 공지 기술을 사용하고, 목적하는 항원 또는 목적하는 항원을 발현하는 세포를 감작 항원으로서 사용하여, 이를 통상의 면역방법에 따라 면역하고, 얻어지는 면역세포를 통상의 세포 융합법에 의해 공지의 친세포와 융합시켜, 통상의 스크리닝법으로 단일클론 항체 생산 세포(하이브리도마)를 스크리닝하고, 얻어진 하이브리도마의 mRNA로부터 역전사효소를 사용하여 항체의 가변영역(V영역)의 cDNA를 합성하여, 이를 목적하는 항체 불변영역(C영역)을 코드하는 DNA와 연결함으로써 얻을 수 있다. A method for obtaining a gene encoding an antibody from a hybridoma is basically using a known technique, using a target antigen or cells expressing the target antigen as a sensitizing antigen, and immunizing it according to a conventional immunization method. , The obtained immune cells are fused with a known parent cell by a conventional cell fusion method, monoclonal antibody-producing cells (hybridoma) are screened by a conventional screening method, and reverse transcriptase is used from the obtained hybridoma mRNA It can be obtained by synthesizing cDNA of the variable region (region V) of an antibody and ligating it with DNA encoding the constant region (region C) of the desired antibody.

보다 구체적으로는, 특별히 아래의 예시에 한정되는 것은 아니지만, 상기 H쇄 및 L쇄를 코드하는 항체 유전자를 얻기 위한 감작항원은 면역원성을 갖는 완전 항원과 면역원성을 나타내지 않는 합텐 등을 포함하는 불완전 항원 양쪽을 포함한다. 예를 들면 목적 단백질의 전장 단백질 또는 부분 펩티드 등을 사용할 수 있다. 그 밖에, 다당류, 핵산, 지질 등으로 구성되는 물질이 항원이 될 수 있는 것이 알려져 있고, 본 발명의 항체의 항원은 특별히 한정되는 것은 아니다. 항원의 조제는 당업자에게 공지의 방법으로 행할 수 있고, 예를 들면 배큘로바이러스를 사용한 방법(예를 들면 WO98/46777 등) 등에 준하여 행할 수 있다. 하이브리도마의 제작은, 예를 들면 밀스테인 등의 방법(G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46) 등에 준하여 행할 수 있다. 항원의 면역원성이 낮은 경우에는, 알부민 등의 면역원성을 갖는 거대분자와 결합시켜 면역을 행하면 된다. 또한 필요에 따라 항원을 다른 분자와 결합시킴으로써 가용성 항원으로 하는 것도 가능하다. 수용체와 같은 막관통 분자를 항원으로서 사용하는 경우, 수용체의 세포외영역 부분을 단편으로서 사용하거나 막관통 분자를 세포 표면 상에 발현하는 세포를 면역원으로서 사용하는 것도 가능하다. More specifically, although not particularly limited to the examples below, the sensitizing antigens for obtaining the antibody genes encoding the H and L chains include complete antigens with immunogenicity and incomplete antigens including haptens that do not exhibit immunogenicity. Contains both antigens. For example, a full-length protein or a partial peptide of the target protein can be used. In addition, it is known that substances composed of polysaccharides, nucleic acids, lipids, etc. can serve as antigens, and the antigen of the antibody of the present invention is not particularly limited. The antigen can be prepared by a method known to those skilled in the art, for example, according to a method using a baculovirus (eg WO98/46777 etc.). The hybridoma can be produced, for example, according to the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46). When the immunogenicity of the antigen is low, immunization may be performed by binding to a macromolecule having immunogenicity such as albumin. It is also possible to obtain a soluble antigen by binding the antigen to other molecules if necessary. When a transmembrane molecule such as a receptor is used as the antigen, it is also possible to use the extracellular domain portion of the receptor as a fragment or to use a cell expressing the transmembrane molecule on the cell surface as the immunogen.

항체 생산 세포는 전술한 적당한 감작항원을 사용하여 동물을 면역화함으로써 얻을 수 있다. 또는 항체를 생산할 수 있는 림프구를 인 비트로(in vitro)에서 면역화하여 항체 생산 세포로 하는 것도 가능하다. 면역화하는 동물로서는 각종 포유동물을 사용할 수 있는데, 설치목, 토끼목, 영장목의 동물이 일반적으로 사용된다. 마우스, 랫트, 햄스터 등의 설치목, 토끼 등의 토끼목, 게잡이원숭이, 빨간털원숭이, 망토비비, 침팬지 등의 원숭이 등의 영장목의 동물을 예시할 수 있다. 그 밖에, 인간 항체 유전자의 레퍼토리를 갖는 형질전환 동물도 알려져 있어, 이러한 동물을 사용함으로써 인간 항체를 얻는 것도 가능하다(WO96/34096; Mendez et al., Nat. Genet. 1997, 15: 146-56 참조). 이러한 형질전환 동물의 사용 대신에, 예를 들면 인간 림프구를 in vitro에서 목적하는 항원 또는 목적하는 항원을 발현하는 세포로 감작하고, 감작 림프구를 인간 골수종 세포(myeloma cells), 예를 들면 U266과 융합시킴으로써 항원으로의 결합 활성을 갖는 목적하는 인간 항체를 얻는 것도 가능하다(일본국 특허공고 평1-59878호 공보 참조). 또한 인간 항체 유전자의 모든 레퍼토리를 갖는 형질전환 동물을 목적하는 항원으로 면역함으로써 목적하는 인간 항체를 취득할 수 있다(WO93/12227, WO92/03918, WO94/02602, WO96/34096, WO96/33735 참조).Antibody-producing cells can be obtained by immunizing an animal with an appropriate sensitizing antigen as described above. Alternatively, it is also possible to immunize a lymphocyte capable of producing an antibody in vitro to obtain an antibody-producing cell. Various mammals can be used as the animal to be immunized, and animals of the Order Rodent, Lemur, and Primate are generally used. Primates, such as rodents, such as rodents, such as a mouse, a rat, and a hamster; In addition, transgenic animals having a repertoire of human antibody genes are also known, and it is possible to obtain human antibodies by using such animals (WO96/34096; Mendez et al., Nat. Genet. 1997, 15: 146-56). Reference). Instead of using such transgenic animals, for example, human lymphocytes are sensitized with the desired antigen or cells expressing the desired antigen in vitro, and the sensitized lymphocytes are fused with human myeloma cells, such as U266. It is also possible to obtain a desired human antibody having antigen-binding activity by doing so (refer to Japanese Patent Application Laid-Open No. 1-59878). In addition, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (see WO93/12227, WO92/03918, WO94/02602, WO96/34096, and WO96/33735). .

동물의 면역화는, 예를 들면 감작항원을 인산염 완충액(Phosphate-Buffered Saline; PBS) 또는 생리식염수 등으로 적절히 희석, 현탁하고, 필요에 따라 애쥬번트를 혼합하여 유화한 후, 동물의 복강 내 또는 피하에 주사함으로써 행해진다. 그 후, 바람직하게는 프로인트 불완전 애쥬번트에 혼합한 감작항원을 4~21일마다 수 회 투여한다. 항체 생산의 확인은 동물 혈청 중의 목적으로 하는 항체 역가를 관용의 방법으로 측정함으로써 행해질 수 있다. Immunization of animals is performed, for example, by appropriately diluting and suspending the sensitizing antigen in Phosphate-Buffered Saline (PBS) or physiological saline, and emulsifying by mixing with an adjuvant if necessary, then intraperitoneally or subcutaneously in the animal. This is done by injecting into After that, the sensitizing antigen mixed with Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. Confirmation of antibody production can be performed by measuring the titer of the target antibody in animal serum by a conventional method.

하이브리도마는 목적하는 항원으로 면역화한 동물 또는 림프구로부터 얻어진 항체 생산 세포를 관용의 융합제(예를 들면 폴리에틸렌글리콜)를 사용하여 골수종 세포와 융합하여 제작할 수 있다(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). 필요에 따라 하이브리도마 세포를 배양·증식하고, 면역침강, 방사면역분석(RIA), 효소결합 면역흡착 분석(ELISA) 등의 공지의 분석법에 의해 상기 하이브리도마로부터 생산되는 항체의 결합 특이성을 측정한다. 그 후, 필요에 따라 목적으로 하는 특이성, 친화성 또는 활성이 측정된 항체를 생산하는 하이브리도마를 한계희석법 등의 수법으로 서브클로닝하는 것도 가능하다. Hybridomas can be prepared by fusing antibody-producing cells obtained from an animal or lymphocytes immunized with a desired antigen with myeloma cells using a conventional fusion agent (eg, polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice). , Academic Press, 1986, 59-103). If necessary, hybridoma cells are cultured and propagated, and the binding specificity of the antibody produced from the hybridoma is tested by known assays such as immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). measure Thereafter, if necessary, it is also possible to subcloning a hybridoma producing an antibody whose specificity, affinity, or activity of interest is measured by a method such as limiting dilution.

계속해서, 선택된 항체를 코드하는 유전자를 하이브리도마 또는 항체 생산 세포(감작 림프구 등)로부터 항체에 특이적으로 결합 가능한 프로브(예를 들면 항체 불변영역을 코드하는 서열에 상보적인 올리고 뉴클레오티드 등)를 사용하여 클로닝할 수 있다. 또한 mRNA로부터 RT-PCR에 의해 클로닝하는 것도 가능하다. 면역글로불린은 IgA, IgD, IgE, IgG 및 IgM의 5개의 다른 클래스로 분류된다. 또한 이들 클래스는 몇 개의 서브클래스(아이소타입)(예를 들면 IgG-1, IgG-2, IgG-3 및 IgG-4;IgA-1 및 IgA-2 등)로 나뉘어진다. 본 발명에 있어서 항체의 제조에 사용하는 H쇄 및 L쇄는 이들 어느 클래스 및 서브클래스에 속하는 항체에 유래하는 것이어도 되고 특별히 한정되지 않으나, IgG는 특히 바람직한 것이다.Subsequently, a probe capable of specifically binding to an antibody (eg, an oligonucleotide complementary to a sequence encoding an antibody constant region, etc.) from a hybridoma or antibody-producing cell (sensitized lymphocyte, etc.) can be used for cloning. It is also possible to clone from mRNA by RT-PCR. Immunoglobulins are classified into five different classes: IgA, IgD, IgE, IgG and IgM. These classes are also divided into several subclasses (isotypes) (eg, IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2, etc.). The H chain and L chain used in the production of the antibody in the present invention may be derived from an antibody belonging to any of these classes and subclasses, and are not particularly limited, but IgG is particularly preferred.

여기서 H쇄 및 L쇄를 코드하는 유전자를 유전자 공학적 수법으로 개변하는 것도 가능하다. 예를 들면 마우스 항체, 랫트 항체, 토끼 항체, 햄스터 항체, 양 항체, 낙타 항체 등의 항체에 대해서 인간에 대한 이종 항원성을 저하시키는 것 등을 목적으로 인위적으로 개변한 유전자 재조합형 항체, 예를 들면 키메라 항체, 인간화 항체 등을 적절히 제작할 수 있다. 키메라 항체는 인간 이외의 포유동물, 예를 들면 마우스 항체의 H쇄, L쇄의 가변영역과 인간 항체의 H쇄, L쇄의 불변영역으로 이루어지는 항체로, 마우스 항체의 가변영역을 코드하는 DNA를 인간 항체의 불변영역을 코드하는 DNA와 연결하고, 이를 발현 벡터에 삽입하고 숙주에 도입하여 생산시킴으로써 얻을 수 있다. 인간화 항체는 재구성(reshaped) 인간 항체로도 칭해지고, 인간 이외의 포유동물, 예를 들면 마우스 항체의 상보성 결정영역(CDR; complementary determining region)을 연결하도록 설계한 DNA 서열을, 말단부에 오버랩되는 부분을 갖도록 제작한 여러 개의 올리고 뉴클레오티드로부터 PCR법에 의해 합성한다. 얻어진 DNA를 인간 항체 불변영역을 코드하는 DNA와 연결하고, 이어서 발현 벡터에 삽입하고 이를 수주에 도입하여 생산시킴으로써 얻어진다(EP239400; WO96/02576 참조). CDR을 매개로 연결되는 인간 항체의 FR은 상보성 결정영역이 양호한 항원 결합 부위를 형성하는 것이 선택된다. 필요에 따라 재구성 인간 항체의 상보성 결정영역이 적절한 항원 결합 부위를 형성하도록 항체 가변영역의 프레임워크 영역의 아미노산을 치환해도 된다(K. Sato et al., Cancer Res. 1993, 53: 851-856). Here, it is also possible to alter the genes encoding the H chain and the L chain by genetic engineering techniques. For example, a genetically modified antibody artificially modified for the purpose of reducing heterologous antigenicity to humans against antibodies such as mouse antibody, rat antibody, rabbit antibody, hamster antibody, sheep antibody, and camel antibody, for example, For example, a chimeric antibody, a humanized antibody, etc. can be prepared suitably. A chimeric antibody is an antibody comprising the variable regions of the H and L chains of a mammal other than human, for example, a mouse antibody and the constant regions of the H and L chains of a human antibody, and comprises DNA encoding the variable region of a mouse antibody. It can be obtained by linking with DNA encoding the constant region of a human antibody, inserting it into an expression vector, introducing it into a host, and producing it. A humanized antibody is also referred to as a reshaped human antibody, and includes a DNA sequence designed to link the complementary determining region (CDR) of a non-human mammal, for example, a mouse antibody, with a portion overlapping at the end. It is synthesized by PCR method from several oligonucleotides prepared to have It is obtained by ligating the obtained DNA with DNA encoding a human antibody constant region, then inserting it into an expression vector, introducing it into an expression vector, and producing it (see EP239400; WO96/02576). Human antibody FRs linked via CDRs are selected to form an antigen-binding site with a good complementarity determining region. If necessary, amino acids in the framework region of the antibody variable region may be substituted so that the complementarity determining region of the reconstituted human antibody forms an appropriate antigen-binding site (K. Sato et al., Cancer Res. 1993, 53: 851-856). .

전술한 인간화 이외에, 예를 들면 항원과의 결합성 등의 항체의 생물학적 특성을 개선하기 위해 개변을 행하는 것도 생각된다. 이러한 개변은 부위 특이적 돌연변이(예를 들면 Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488 참조), PCR 변이, 카세트 변이 등의 방법으로 행할 수 있다. 일반적으로 생물학적 특성이 개선된 항체 변이체는 70% 이상, 보다 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상(예를 들면 95% 이상, 97%, 98%, 99% 등)의 아미노산 서열 상동성 및/또는 유사성을 기초가 된 항체 가변영역의 아미노산 서열에 대해 갖는다. 본 명세서에 있어서 서열의 상동성 및/또는 유사성은 서열 상동성이 최대값을 취하도록 필요에 따라 서열을 정렬화 및 갭 도입한 후, 기초가 된 항체 잔기와 상동(동일한 잔기) 또는 유사(일반적인 아미노산 측쇄의 특성에 기초하여 동일한 그룹으로 분류되는 아미노산 잔기)한 아미노산 잔기의 비율로서 정의된다. 통상 천연의 아미노산 잔기는 그 측쇄의 성질에 기초하여 (1)소수성：알라닌, 이소류신, 노르류신, 발린, 메티오닌 및 류신；(2)중성 친수성：아스파라긴, 글루타민, 시스테인, 트레오닌 및 세린；(3)산성：아스파라긴산 및 글루타민산；(4)염기성：아르기닌, 히스티딘 및 리신；(5)사슬의 배향에 영향을 미치는 잔기：글리신 및 프롤린；및 (6)방향족성：티로신, 트립토판 및 페닐알라닌의 그룹으로 분류된다. In addition to the humanization described above, it is also conceivable to modify the antibody in order to improve the biological properties of the antibody, such as binding to an antigen, for example. Such modifications can be performed by methods such as site-specific mutagenesis (see, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutation, and cassette mutation. In general, the antibody variant with improved biological properties has an amino acid sequence of 70% or more, more preferably 80% or more, even more preferably 90% or more (eg 95% or more, 97%, 98%, 99%, etc.) with respect to the amino acid sequence of the antibody variable region on the basis of homology and/or similarity. The homology and/or similarity of sequences herein refers to homology (identical residues) or similar (general residues) to the antibody residues on which they are based, after aligning the sequences and introducing gaps as necessary so that the sequence homology takes the maximum value. It is defined as the proportion of one amino acid residue (amino acid residues classified into the same group based on the properties of the amino acid side chain). Usually, natural amino acid residues are based on the nature of their side chains, (1) hydrophobicity: alanine, isoleucine, norleucine, valine, methionine and leucine; (2) neutral hydrophilicity: asparagine, glutamine, cysteine, threonine and serine; (3) Acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine and lysine; (5) residues affecting chain orientation: glycine and proline; and (6) aromatic: tyrosine, tryptophan and phenylalanine. .

통상, H쇄 및 L쇄의 가변영역 중에 존재하는 전부 6개의 상보성 결정영역(초가변부;CDR)이 상호작용하여 항체의 항원 결합 부위를 형성하고 있다. 이 중 하나의 가변영역이라도 전체 결합 부위를 포함하는 것보다는 낮은 친화성이 되지만, 항원을 인식하고 결합하는 능력이 있는 것이 알려져 있다. 따라서 본 발명의 H쇄 및 L쇄를 코드하는 항체 유전자는 그 유전자에 의해 코드되는 폴리펩티드가 목적하는 항원과의 결합성을 유지하고 있으면 되고, H쇄 및 L쇄의 각각의 항원 결합 부위를 포함하는 단편 부분을 코드하고 있으면 된다. In general, all six complementarity determining regions (hypervariable regions; CDRs) present in the variable regions of the H and L chains interact to form an antigen-binding site of the antibody. Even one of these variable regions has a lower affinity than that containing the entire binding site, but is known to have the ability to recognize and bind antigen. Therefore, the antibody gene encoding the H chain and L chain of the present invention only needs to maintain binding to the desired antigen by the polypeptide encoded by the gene, and contains antigen-binding sites of the H chain and L chain, respectively. You just need to code the fragment part.

폴리펩티드의 활성 및 항원의 예시Examples of Polypeptide Activity and Antigen

본 발명의 해리 및/또는 회합 제어방법을 이용함으로써, 예를 들면 활성을 갖는 항체 또는 폴리펩티드를 효율적으로 제작할 수 있다. 상기 활성으로서는, 예를 들면 결합 활성, 중화 활성, 세포 상해 활성, 아고니스트 활성, 안타고니스트 활성, 효소 활성 등을 들 수 있다. 아고니스트 활성이란, 수용체 등의 항원에 항체가 결합함으로써 세포 내에 시그널이 전달되거나 하여 어떠한 생리학적 활성의 변화를 유도하는 활성이다. 생리학적 활성으로서는, 예를 들면 증식 활성, 생존 활성, 분화 활성, 전사 활성, 막 수송 활성, 결합 활성, 단백질 분해 활성, 인산화/탈인산화 활성, 산화 환원 활성, 전이 활성, 핵산 분해 활성, 탈수 활성, 세포사 유도 활성, 아포토시스 유도 활성을 들 수 있는데, 이들에 한정되지 않는다. By using the dissociation and/or association control method of the present invention, for example, an antibody or polypeptide having an activity can be efficiently produced. Examples of the activity include binding activity, neutralizing activity, cytotoxic activity, agonist activity, antagonist activity, and enzyme activity. The agonist activity is an activity that induces a change in a certain physiological activity by transmitting a signal into a cell by binding of an antibody to an antigen such as a receptor. The physiological activity includes, for example, proliferation activity, survival activity, differentiation activity, transcriptional activity, membrane transport activity, binding activity, proteolytic activity, phosphorylation/dephosphorylation activity, redox activity, transfer activity, nucleic acid degradation activity, dehydration activity. , cell death-inducing activity, and apoptosis-inducing activity, but are not limited thereto.

또한 본 발명의 방법에 의해 목적하는 항원을 인식하거나 또는 목적하는 수용체와 결합하는 항체 또는 폴리펩티드를 효율적으로 제작할 수 있다. In addition, by the method of the present invention, an antibody or polypeptide that recognizes a target antigen or binds to a target receptor can be efficiently produced.

본 명세서에 있어서 항원은 특별히 한정되지 않고, 어떠한 항원이어도 된다. 항원의 예로서는, 예를 들면 리간드(사이토카인, 케모카인 등), 수용체, 암 항원, MHC 항원, 분화 항원, 면역글로불린 및 면역글로불린을 일부에 포함하는 면역 복합체를 적합하게 들 수 있다. The antigen in this specification is not specifically limited, Any antigen may be sufficient. Suitable examples of the antigen include, for example, ligands (cytokines, chemokines, etc.), receptors, cancer antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes partially including immunoglobulins.

사이토카인의 예로서는, 인터류킨 1~18, 콜로니 자극인자(G-CSF, M-CSF, GM-CSF 등), 인터페론(IFN-α, IFN-β, IFN-γ 등), 성장인자(EGF, FGF, IGF, NGF, PDGF, TGF, HGF 등), 종양 괴사 인자(TNF-α, TNF-β), 림포톡신, 에리트로포이에틴, 렙틴, SCF, TPO, MCAF, BMP를 들 수 있다.Examples of cytokines include interleukins 1 to 18, colony stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN-α, IFN-β, IFN-γ, etc.), growth factors (EGF, FGF, etc.) , IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factor (TNF-α, TNF-β), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF, and BMP.

케모카인의 예로서는, CCL1~CCL28 등의 CC 케모카인, CXCL1~CXCL17 등의 CXC 케모카인, XCL1~XCL2 등의 C 케모카인, CX3CL1 등의 CX3C 케모카인을 들 수 있다. Examples of the chemokines include CC chemokines such as CCL1-CCL28, CXC chemokines such as CXCL1-CXCL17, C chemokines such as XCL1-XCL2, and CX3C chemokines such as CX3CL1.

수용체의 예로서는, 예를 들면 조혈 인자 수용체 패밀리, 사이토카인 수용체 패밀리, 티로신키나아제형 수용체 패밀리, 세린/트레오닌키나아제형 수용체 패밀리, TNF 수용체 패밀리, G 단백질 공액형 수용체 패밀리, GPI 앵커형 수용체 패밀리, 티로신포스파타아제형 수용체 패밀리, 접착인자 패밀리, 호르몬 수용체 패밀리 등의 수용체 패밀리에 속하는 수용체 등을 들 수 있다. 이들 수용체 패밀리에 속하는 수용체 및 그 특징에 관해서는 다수의 문헌, 예를 들면 Cooke BA., King RJB., van der Molen HJ. ed. New Comprehesive Biochemistry Vol.18B "Hormones and their Actions Part II"pp.1-46 (1988) Elsevier Science Publishers BV., Patthy(Cell (1990) 61 (1), 13-14), Ullrich 등(Cell (1990) 61 (2), 203-212), Massague(e에는 아큐트 악센트 기호가 붙는다)(Cell (1992) 69 (6), 1067-1070), Miyajima 등(Annu. Rev. Immunol. (1992) 10, 295-331), Taga 등(FASEB J. (1992) 6, 3387-3396), Fantl 등(Annu. Rev. Biochem. (1993), 62, 453-481), Smith 등(Cell (1994) 76 (6) 959-962), Flower DR.(Biochim. Biophys. Acta (1999) 1422 (3) 207-234) 등에 기재되어 있다. Examples of the receptor include, for example, hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine/threonine kinase type receptor family, TNF receptor family, G protein conjugated receptor family, GPI anchor type receptor family, tyrosine phosphatase. and receptors belonging to receptor families such as the patase-type receptor family, the adhesion factor family, and the hormone receptor family. Receptors belonging to these receptor families and their characteristics are discussed in numerous literatures, for example Cooke BA., King RJB., van der Molen HJ. ed. New Comprehesive Biochemistry Vol.18B "Hormones and their Actions Part II" pp.1-46 (1988) Elsevier Science Publishers BV., Patthy (Cell (1990) 61 (1), 13-14), Ullrich et al. (Cell (1990) ) 61 (2), 203-212), Massague (e is accented with an accusative) (Cell (1992) 69 (6), 1067-1070), Miyajima et al. (Annu. Rev. Immunol. (1992) 10) , 295-331), Taga et al. (FASEB J. (1992) 6, 3387-3396), Fantl et al. (Annu. Rev. Biochem. (1993), 62, 453-481), Smith et al. (Cell (1994) 76) (6) 959-962), Flower DR. (Biochim. Biophys. Acta (1999) 1422 (3) 207-234) and the like.

상기 수용체 패밀리에 속하는 구체적인 수용체로서는, 예를 들면 인간 또는 마우스 에리트로포이에틴(EPO) 수용체(Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277-285), 인간 또는 마우스 과립구 콜로니 자극인자(G-CSF) 수용체(Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341-350), 인간 또는 마우스 트롬보포이에틴(TPO) 수용체(Proc Natl Acad Sci U S A. (1992) 89 (12), 5640-5644, EMBO J. (1993) 12(7), 2645-53), 인간 또는 마우스 인슐린 수용체(Nature (1985) 313 (6005), 756-761), 인간 또는 마우스 Flt-3 리간드 수용체(Proc. Natl. Acad. Sci. USA. (1994) 91 (2), 459-463), 인간 또는 마우스 혈소판 유래 증식인자(PDGF) 수용체(Proc. Natl. Acad. Sci. USA. (1988) 85 (10) 3435-3439), 인간 또는 마우스 인터페론(IFN)-α, β수용체(Cell (1990) 60 (2), 225-234. 및 Cell (1994) 77 (3), 391-400), 인간 또는 마우스 렙틴 수용체, 인간 또는 마우스 성장 호르몬(GH) 수용체, 인간 또는 마우스 인터류킨(IL)-10 수용체, 인간 또는 마우스 인슐린 증식인자(IGF)-I 수용체, 인간 또는 마우스 백혈병 억제인자(LIF) 수용체, 인간 또는 마우스 모양체 신경영양인자(CNTF) 수용체 등이 적합하게 예시된다.Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptor (Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277-285); Human or mouse granulocyte colony stimulating factor (G-CSF) receptor (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341) -350), human or mouse thrombopoietin (TPO) receptor (Proc Natl Acad Sci US A. (1992) 89 (12), 5640-5644, EMBO J. (1993) 12(7), 2645-53) , human or mouse insulin receptor (Nature (1985) 313 (6005), 756-761), human or mouse Flt-3 ligand receptor (Proc. Natl. Acad. Sci. USA. (1994) 91 (2), 459- 463), human or mouse platelet-derived proliferation factor (PDGF) receptor (Proc. Natl. Acad. Sci. USA. (1988) 85 (10) 3435-3439), human or mouse interferon (IFN)-α, β receptor ( Cell (1990) 60 (2), 225-234. and Cell (1994) 77 (3), 391-400), human or mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL) )-10 receptor, human or mouse insulin growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human or mouse ciliary body neurotrophic factor (CNTF) receptor, etc. are exemplified suitably.

암 항원은 세포의 악성화에 수반하여 발현되는 항원으로, 종양 특이성 항원으로도 불린다. 또한 세포가 암화되었을 때 세포 표면이나 단백질 분자 상에 나타나는 이상한 당쇄도 암 항원으로, 암 당쇄 항원으로도 불린다. 암 항원의 예로서는, 예를 들면 상기 수용체로서 GPI 앵커형 수용체 패밀리에 속하는 간암을 비롯한 몇 가지 암에 있어서 발현되고 있는 GPC3(Int J Cancer. (2003) 103 (4), 455-65), 폐암을 비롯한 복수의 암에서 발현되는 EpCAM(Proc Natl Acad Sci U S A. (1989) 86 (1), 27-31), CA19-9, CA15-3, 시리얼 SSEA-1(SLX) 등을 적합하게 들 수 있다. Cancer antigens are antigens expressed with malignancy of cells, and are also called tumor-specific antigens. Also, abnormal sugar chains that appear on the cell surface or protein molecules when cells are cancerous are also called cancer antigens and cancer sugar chain antigens. Examples of the cancer antigen include GPC3 (Int J Cancer. (2003) 103 (4), 455-65) expressed in several cancers including liver cancer belonging to the GPI anchor type receptor family as the receptor, and lung cancer. Suitable examples include EpCAM (Proc Natl Acad Sci US A. (1989) 86 (1), 27-31), CA19-9, CA15-3, serial SSEA-1 (SLX), etc. have.

MHC 항원은 주로 MHC class I 항원과 MHC class II 항원으로 분류되고, MHC class I 항원에는 HLA-A, -B, -C, -E, -F, -G, -H가 포함되며, MHC class II 항원에는 HLA-DR, -DQ, -DP가 포함된다. MHC antigens are mainly classified into MHC class I antigens and MHC class II antigens, and MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, -H, and MHC class II antigens. Antigens include HLA-DR, -DQ, -DP.

분화 항원에는 CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, CDw130이 포함될 수 있다.Differentiation antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30 , CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56 , CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, CDw130.

면역글로불린에는 IgA, IgM, IgD, IgG, IgE가 포함된다. 또한 면역 복합체는 적어도 면역글로불린 중 어느 하나의 성분을 포함한다.Immunoglobulins include IgA, IgM, IgD, IgG, and IgE. In addition, the immune complex includes at least one component of immunoglobulin.

기타 항원으로서는 하기와 같은 분자；17-IA, 4-1BB, 4Dc, 6-케토-PGF1a, 8-이소-PGF2a, 8-옥소-dG, A1 아데노신 수용체, A33, ACE, ACE-2, 액티빈, 액티빈 A, 액티빈 AB, 액티빈 B, 액티빈 C, 액티빈 RIA, 액티빈 RIA ALK-2, 액티빈 RIB ALK-4, 액티빈 RIIA, 액티빈 RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, 어드레신, aFGF, ALCAM, ALK, ALK-1, ALK-7, 알파-1-안티트립신, 알파-V/베타-1안타고니스트, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, 아르테민, 항Id, ASPARTIC, 심방성 나트륨 이뇨인자, av/b3 인테그린, Axl, b2M, B7-1, B7-2, B7-H, B-림프구 자극인자(BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3(오스테오게닌(Osteogenin)), BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB(ALK-6), BRK-2, RPK-1, BMPR-II(BRK-3), BMP, b-NGF, BOK, 봄베신, 뼈 유래 신경영양인자, BPDE, BPDE-DNA, BTC, 보체인자3(C3), C3a, C4, C10, CA125, CAD-8, 칼시토닌, cAMP, 암 태아성 항원(CEA), 암 관련 항원, 카텝신 A, 카텝신 B, 카텝신 C/DPPI, 카텝신 D, 카텝신 E, 카텝신 H, 카텝신 L, 카텝신 O, 카텝신 S, 카텝신 V, 카텝신 X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33(p67 단백질), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, 보툴리눔균 독소, 웰치균 독소, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, 사이토케라틴 종양 관련 항원, DAN, DCC, DcR3, DC-SIGN, 보체 제어인자(Decay accelerating factor), des(1-3)-IGF-I(뇌IGF-1), Dhh, 디곡신, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, 엔도텔린 수용체, 엔도케팔리나아제, eNOS, Eot, 에오탁신 1, EpCAM, 에프린 B2/EphB4, EPO, ERCC, E-셀렉틴, ET-1, 팩터 IIa, 팩터 VII, 팩터 VIIIc, 팩터 IX, 섬유아세포 활성화 단백질(FAP), Fas, FcR1, FEN-1, 페리틴, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, 피브린, FL, FLIP, Flt-3, Flt-4, 난포 자극 호르몬, 프랙탈카인, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3(Vgr-2), GDF-5(BMP-14, CDMP-1), GDF-6(BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8(미오스타틴), GDF-9, GDF-15(MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-알파1, GFR-알파2, GFR-알파3, GITR, 글루카곤, Glut4, 당단백질 IIb/IIIa(GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, 성장 호르몬 방출인자, 합텐(NP-cap 또는 NIP-cap), HB-EGF, HCC, HCMV gB 엔벨로프 당단백질, HCMV gH 엔벨로프 당단백질, HCMV UL, 조혈 성장인자(HGF), Hep B gp120, 헤파라나아제, Her2, Her2/neu(ErbB-2), Her3(ErbB-3), Her4(ErbB-4), 단순 헤르페스 바이러스(HSV) gB 당단백질, HSV gD 당단백질, HGFA, 고분자량 흑색종 관련 항원(HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 루프, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, 인간 심장 미오신, 인간 사이토메갈로바이러스(HCMV), 인간 성장 호르몬(HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA 수용체, IgE, IGF, IGF 결합 단백질, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, 인터페론(INF)-알파, INF-베타, INF-감마, 인히빈, iNOS, 인슐린 A쇄, 인슐린 B쇄, 인슐린 유사 증식인자 1, 인테그린 알파 2, 인테그린 알파 3, 인테그린 알파 4, 인테그린 알파 4/베타 1, 인테그린 알파 4/베타 7, 인테그린 알파 5(알파 V), 인테그린 알파 5/베타 1, 인테그린 알파 5/베타 3, 인테그린 알파 6, 인테그린 베타 1, 인테그린 베타 2, 인터페론감마, IP-10, I-TAC, JE, 칼리크레인 2, 칼리크레인 5, 칼리크레인 6, 칼리크레인 11, 칼리크레인 12, 칼리크레인 14, 칼리크레인 15, 칼리크레인 L1, 칼리크레인 L2, 칼리크레인 L3, 칼리크레인 L4, KC, KDR, 케라티노사이트 증식인자(KGF), 라미닌 5, LAMP, LAP, LAP(TGF-1), 잠재적 TGF-1, 잠재적 TGF-1 bp1, LBP, LDGF, LECT2, 레프티, 루이스-Y 항원, 루이스-Y 관련 항원, LFA-1, LFA-3, Lfo, LIF, LIGHT, 리포단백질, LIX, LKN, Lptn, L-셀렉틴, LT-a, LT-b, LTB4, LTBP-1, 폐 표면, 황체 형성 호르몬, 림포톡신 베타 수용체, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, 메탈로프로테아제, MGDF 수용체, MGMT, MHC(HLA-DR), MIF, MIG, MIP, MIP-1-알파, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, 뮤틴(Muc1), MUC18, 뮐러관 억제물질, Mug, MuSK, NAIP, NAP, NCAD, N-C 어드헤린, NCA 90, NCAM, NCAM, 네프릴리신, 뉴로트로핀-3, -4, 또는 -6, 뉴투린, 신경 성장인자(NGF), NGFR, NGF-베타, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, 부갑상선 호르몬, PARC, PARP, PBR, PBSF, PCAD, P-카드헤린, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, 태반성 알칼리포스파타아제(PLAP), PlGF, PLP, PP14, 프로인슐린, 프로릴랙신, 프로테인 C, PS, PSA, PSCA, 전립선 특이적 막 항원(PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, 릴랙신 A쇄, 릴랙신 B쇄, 레닌, 호흡기 다핵체 바이러스(RSV)F, RSV Fgp, Ret, 류머티즘 인자, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, 혈청 알부민, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72(종양 관련 당단백질-72), TARC, TCA-3, T세포 수용체(예를 들면 T세포 수용체 알파/베타), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, 고환 PLAP 유사 알칼리포스파타아제, TfR, TGF, TGF-알파, TGF-베타, TGF-베타 Pan Specific, TGF-베타 RI(ALK-5), TGF-베타 RII, TGF-베타 RIIb, TGF-베타 RIII, TGF-베타 1, TGF-베타 2, TGF-베타 3, TGF-베타 4, TGF-베타 5, 트롬빈, 흉선 Ck-1, 갑상선 자극 호르몬, Tie, TIMP, TIQ, 조직인자, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-알파, TNF-알파베타, TNF-베타 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1 Apo-2, DR4), TNFRSF10B(TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcR1, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 리간드, TL2), TNFSF11(TRANCE/RANK 리간드 ODF, OPG 리간드), TNFSF12(TWEAK Apo-3 리간드, DR3 리간드), TNFSF13(APRIL TALL2), TNFSF13B(BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM 리간드, LTg), TNFSF15(TL1A/VEGI), TNFSF18(GITR 리간드 AITR 리간드, TL6), TNFSF1A(TNF-a 코넥틴(Conectin), DIF, TNFSF2), TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb TNFC, p33), TNFSF4(OX40 리간드 gp34, TXGP1), TNFSF5(CD40 리간드 CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6(Fas 리간드 Apo-1 리간드, APT1 리간드), TNFSF7(CD27 리간드 CD70), TNFSF8(CD30 리간드 CD153), TNFSF9(4-1BB 리간드 CD137 리간드), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, 트랜스페린 수용체, TRF, Trk, TROP-2, TSG, TSLP, 종양 관련 항원 CA125, 종양 관련 항원 발현 루이스 Y 관련 탄수화물, TWEAK, TXB2, Ung, uPAR, uPAR-1, 우로키나아제, VCAM, VCAM-1, VECAD, VE-카드헤린, VE-카드헤린-2, VEFGR-1(flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, 바이러스 항원, VLA, VLA-1, VLA-4, VNR 인테그린, 폰 빌레브란트 인자, WIF-1, WNT1, WNT2, WNT2B／13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17/IL-17R, IL-20/IL-20R, 산화 LDL, PCSK9, 프리칼리크레인, RON, TMEM16F, SOD1, 크로모그라닌 A, 크로모그라닌 B, tau, VAP1, 고분자 키니노겐, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, 팩터 B, 팩터 D, 팩터 H, 프로페르딘, 스클레로스틴, 피브리노겐, 피브린, 프로트롬빈, 트롬빈, 조직인자, 팩터 V, 팩터 Va, 팩터 VII, 팩터 VIIa, 팩터 VIII, 팩터 VIIIa, 팩터 IX, 팩터 IXa, 팩터 X, 팩터 Xa, 팩터 XI, 팩터 XIa, 팩터 XII, 팩터 XIIa, 팩터 XIII, 팩터 XIIIa, TFPI, 안티트롬빈 III, EPCR, 트롬보모듈린, TAPI, tPA, 플라스미노겐, 플라스민, PAI-1, PAI-2, GPC3, 신데칸-1, 신데칸-2, 신데칸-3, 신데칸-4, LPA, S1P, 호르몬 및 성장인자를 위한 수용체가 예시될 수 있다.Other antigens include the following molecules; 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin , activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15 , ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2 , B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b- ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 (Osteogenin), BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK -2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4 , C10, CA125, CAD-8, calcitonin, cAMP, cancer fetal antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H , cathepsin L, cathepsin O, cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 ( B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Welch toxin, CKb8-1, CLC, CMV , CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin Tumor-associated antigens, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, endokephalinase, eNOS, Eot , Eotaxin 1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-Selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast Activating Protein (FAP), Fas, FcR1, FEN -1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3(Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF- 15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM -CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor ( HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4) ), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24 , HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg , Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4/beta 1, integrin alpha 4/beta 7, integrin alpha 5 (alpha V), integrin alpha 5/beta 1, integrin alpha 5/beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, Interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein Crane L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Potential TGF-1, Potential TGF-1 bp1, LBP, LDGF, LECT2 , Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, L T-b, LTB4, LTBP-1, Lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, Metalloproteases, MGDF receptors, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mutin (Muc1), MUC18 , Mullerian tube inhibitors, Mug, MuSK, NAIP, NAP, NCAD, N-C adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, Nuturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC , PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP) ), PlGF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, Prostate Specific Membrane Antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES , relaxin A chain, relaxin B chain, renin, respiratory polynuclear virus (RSV)F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin , sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMD F, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptor (e.g. T-cell receptor alpha/ beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK- 5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymus Ck-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo -2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (TRANCE ODF) R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF16 (NGFR p75NTR) BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRSF3), TNFRSF (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BBF9) CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2) Ligand, TL2), TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT) HVEM ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70) , TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, virus Antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKKsp90, EREG IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, macromolecular kinino Gen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, Factor B, Factor D, Factor H, Properdin , sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, Factor XIa, Factor XII, Factor XIIa, Factor XIII, Factor XIIIa, TFPI, Antithrombin III, EPCR, Thrombomodulin, TAPI, tPA, Plasminogen, Plasmin, PAI-1, PAI-2, GPC3, Neo Deccan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, Receptors for S1P, hormones and growth factors can be exemplified.

본 발명에 있어서의 비한정의 일실시태양에서는, 이중특이성 항체의 하나의 특이성은 암 항원을 표적으로 하고, 다른 쪽의 특이성은 CTL(Cytotoxic T lymphocyte)에 발현하는 항원, 예를 들면 CD3나 TNFRSF(Tumor Necrosis Factor Receptor Super Family)를 표적으로 할 수 있는데, 이 조합에 한정되는 것은 아니다. TNFRSF의 예로서는 TNFRSF9(CD137), TNFRSF5(CD40) 및 TNFRSF4(OX40) 등을 들 수 있다. In one non-limiting embodiment of the present invention, one specificity of the bispecific antibody targets a cancer antigen, and the other specificity is an antigen expressed on CTL (Cytotoxic T lymphocyte), for example, CD3 or TNFRSF. (Tumor Necrosis Factor Receptor Super Family) can be targeted, but is not limited to this combination. Examples of TNFRSF include TNFRSF9 (CD137), TNFRSF5 (CD40) and TNFRSF4 (OX40).

핵산의 개변modification of nucleic acids

또한 본 발명의 제조방법의 다른 태양에 있어서는, 본 발명은 폴리펩티드 간의 해리 및/또는 회합이 제어되도록, 폴리펩티드 간의 계면을 형성하는 아미노산 잔기(예를 들면 EU 넘버링에 의한 356번 위치 및 439번 위치, 357번 위치 및 370번 위치, 및 399번 위치 및 409번 위치의 아미노산 잔기), 및/또는 EU 넘버링 397번 위치 및/또는 392번 위치의 아미노산 잔기에 변이를 갖는 이종 다량체의 제조방법으로서, (a) 폴리펩티드 간의 해리 및 회합이 제어되도록, 당해 폴리펩티드 간의 계면 등을 형성하는 아미노산 잔기를 코드하는 핵산을 원래의 핵산으로부터 개변하는 공정, (b) 당해 폴리펩티드를 발현하도록 당해 핵산을 갖는 숙주세포를 배양하는 공정, (c) 당해 숙주세포의 배양물로부터 당해 폴리펩티드를 회수하는 공정, 및 (d) 환원 조건하, 각 폴리펩티드를 인큐베이트하여 목적하는 폴리펩티드의 헤테로체를 회수하는 공정을 포함하는, 이종 다량체의 제조방법을 제공한다. Further, in another aspect of the production method of the present invention, the present invention provides amino acid residues forming an interface between polypeptides (for example, positions 356 and 439 by EU numbering, A method for preparing a heteromultimer having a mutation in the amino acid residues at positions 357 and 370, and at positions 399 and 409), and/or at the amino acid residues at EU numbering positions 397 and/or 392, (a) a step of modifying a nucleic acid encoding an amino acid residue forming an interface between the polypeptides from the original nucleic acid so as to control dissociation and association between the polypeptides; (b) a host cell having the nucleic acid to express the polypeptide A heterologous heterologous cell comprising the steps of culturing, (c) recovering the polypeptide from the culture of the host cell, and (d) incubating each polypeptide under reducing conditions to recover heteroforms of the desired polypeptide. A method for preparing a multimer is provided.

또한 본 발명의 전술한 해리 및/또는 회합 제어방법을 이용하여 폴리펩티드 간의 회합이 저해되도록, 당해 폴리펩티드 간의 계면을 형성하는 아미노산 잔기를 코드하는 핵산을 원래의 핵산으로부터 개변하는 공정을 포함하는 방법도 또한 본 발명의 상기 제조방법의 바람직한 태양 중 하나이다. Also, a method comprising a step of modifying a nucleic acid encoding an amino acid residue forming an interface between the polypeptides from the original nucleic acid so that the association between the polypeptides is inhibited using the above-described dissociation and/or association control method of the present invention is also provided. It is one of the preferable aspects of the said manufacturing method of this invention.

본 발명의 상기 방법에 있어서 「핵산을 개변한다」는 것은, 본 발명에 있어서의 「개변」에 의해 도입되는 아미노산 잔기에 대응하도록 핵산을 개변하는 것을 말한다. 보다 구체적으로는, 원래의(개변 전의) 아미노산 잔기를 코드하는 핵산에 대해서 개변에 의해 도입되는 아미노산 잔기를 코드하는 핵산으로 개변하는 것을 말한다. 통상, 목적의 아미노산 잔기를 코드하는 코돈이 되도록 원래의 핵산에 대해 적어도 1염기를 삽입, 결실 또는 치환하는 유전자 조작 또는 변이 처리를 행하는 것을 의미한다. 즉, 원래의 아미노산 잔기를 코드하는 코돈은 개변에 의해 도입되는 아미노산 잔기를 코드하는 코돈에 의해 치환된다. 이러한 핵산의 개변은 당업자에게 있어서는 공지의 기술, 예를 들면 부위 특이적 변이 유발법, PCR 변이 도입법 등을 사용하여 적절히 실시하는 것이 가능하다. In the above method of the present invention, "modification of a nucleic acid" refers to altering a nucleic acid so as to correspond to an amino acid residue introduced by "modification" in the present invention. More specifically, it refers to alteration of a nucleic acid encoding an amino acid residue introduced by modification with respect to a nucleic acid encoding the original (before modification) amino acid residue. Usually, it means to perform genetic manipulation or mutation treatment in which at least one base is inserted, deleted or substituted in the original nucleic acid so as to become a codon encoding the desired amino acid residue. That is, the codon encoding the original amino acid residue is substituted by the codon encoding the amino acid residue introduced by the alteration. Such modifications of nucleic acids can be appropriately carried out by those skilled in the art using known techniques, for example, site-specific mutagenesis, PCR mutagenesis, and the like.

또한 본 발명에 있어서의 핵산은 통상 적당한 벡터로 담지(삽입)되어 숙주세포로 도입된다. 상기 벡터로서는 삽입한 핵산을 안정하게 유지하는 것이라면 특별히 제한되지 않고, 예를 들면 숙주로 대장균을 사용하는 것이라면, 클로닝용 벡터로서는 pBluescript 벡터(Stratagene사 제조) 등이 바람직한데 시판의 각종 벡터를 이용할 수 있다. 본 발명의 폴리펩티드를 생산할 목적으로 벡터를 사용하는 경우에는 특히 발현 벡터가 유용하다. 발현 벡터로서는 시험관 내, 대장균 내, 배양세포 내, 생물개체 내에서 폴리펩티드를 발현하는 벡터라면 특별히 제한되지 않는데, 예를 들면 시험관 내 발현이라면 pBEST 벡터(프로메가사 제조), 대장균이라면 pET 벡터(Invitrogen사 제조), 배양세포라면 pME18S-FL3 벡터(GenBank Accession No. AB009864), 생물개체라면 pME18S 벡터(Mol Cell Biol. 8:466-472(1988)) 등이 바람직하다. 벡터로의 본 발명의 DNA의 삽입은 통상의 방법으로, 예를 들면 제한효소 사이트를 사용한 리가아제 반응에 의해 행할 수 있다(Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons．Section 11.4-11.11). In addition, the nucleic acid in the present invention is usually carried (inserted) with an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it stably maintains the inserted nucleic acid. For example, if Escherichia coli is used as the host, pBluescript vector (manufactured by Stratagene), etc. is preferable as the vector for cloning. Various commercially available vectors can be used. have. Expression vectors are particularly useful when vectors are used for the purpose of producing the polypeptides of the present invention. The expression vector is not particularly limited as long as it is a vector expressing the polypeptide in vitro, in E. coli, in cultured cells, or in a living organism. For example, if it is expressed in vitro, pBEST vector (manufactured by Promega), or pET vector (Invitrogen) for in vitro expression. manufacture), the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) for living organisms are preferable. Insertion of the DNA of the present invention into a vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley). & Sons. Sections 11.4-11.11).

상기 숙주세포로서 특별히 제한은 없고, 목적에 따라 각종 숙주세포가 사용된다. 폴리펩티드를 발현시키기 위한 세포로서는, 예를 들면 세균세포(예：스트렙토코커스, 스타필로코커스, 대장균, 스트렙토마이세스, 고초균), 진균세포(예：효모, 아스퍼질러스), 곤충세포(예：드로소필라 S2, 스포도프테라 SF9), 동물세포(예：CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) 및 식물세포를 예시할 수 있다. 숙주세포로의 벡터 도입은, 예를 들면 인산칼슘 침전법, 전기 펄스 천공법(Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons．Section 9.1-9.9), 리포펙타민법(GIBCO-BRL사 제조), 마이크로인젝션법 등의 공지의 방법으로 행하는 것이 가능하다. There is no restriction|limiting in particular as said host cell, According to the objective, various host cells are used. Cells for expressing the polypeptide include, for example, bacterial cells (eg, Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, drosophilus). Sophila S2, Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells can be exemplified. Vector introduction into host cells is, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), Lipofecta It is possible to carry out by a known method such as a civil method (manufactured by GIBCO-BRL) and a microinjection method.

숙주세포에 있어서 발현된 폴리펩티드를 소포체의 내강에, 세포 주유강에, 또는 세포 외의 환경에 분비시키기 위해 적당한 분비 시그널을 목적의 폴리펩티드에 삽입할 수 있다. 이들 시그널은 목적의 폴리펩티드에 대해 내인성이어도 되고 이종 시그널이어도 된다. In order to secrete the polypeptide expressed in the host cell into the lumen of the endoplasmic reticulum, into the peritoneal cavity, or into the extracellular environment, an appropriate secretion signal can be inserted into the target polypeptide. These signals may be endogenous to the target polypeptide or may be heterologous signals.

상기 제조방법에 있어서의 폴리펩티드의 회수는 본 발명의 폴리펩티드가 배지에 분비되는 경우는 배지를 회수한다. 본 발명의 폴리펩티드가 세포 내에 생산되는 경우는, 그 세포를 먼저 용해하고 그 후에 폴리펩티드를 회수한다. For the recovery of the polypeptide in the above production method, the medium is recovered when the polypeptide of the present invention is secreted into the medium. When the polypeptide of the present invention is produced in a cell, the cell is first lysed, and then the polypeptide is recovered.

재조합 세포 배양물로부터 본 발명의 폴리펩티드를 회수하여 정제하는 데는 황산암모늄 또는 에탄올 침전, 산 추출, 음이온 또는 양이온 교환 크로마토그래피, 포스포셀룰로오스 크로마토그래피, 소수성 상호작용 크로마토그래피, 친화성 크로마토그래피, 히드록실아파타이트 크로마토그래피 및 렉틴 크로마토그래피를 포함한 공지의 방법을 사용할 수 있다. For the recovery and purification of the polypeptide of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl Known methods including apatite chromatography and lectin chromatography can be used.

본 발명의 비한정의 실시태양으로서 각각 제1 및 제2 폴리펩티드의 호모체를 생산하는 각 세포주를 배양하고, 당해 배양상청을 정제 후에 정제 항체를 사용하여 FAE(Fab Arm Exchange) 반응을 일으키는 방법, 각각 제1 및 제2 폴리펩티드의 호모체를 생산하는 각 세포주를 배양하고, 당해 배양상청을 정제하지 않고 혼합하여 혼합 배양상청 중에서 FAE 반응을 일으킨 후 정제하는 방법, 제1 폴리펩티드의 호모체를 생산하는 세포주와 제2 폴리펩티드의 호모체를 생산하는 세포주를 혼합하여 배양하고, 당해 배양상청을 정제 후에 정제 항체를 사용하여 FAE 반응을 일으키는 방법, 제1 폴리펩티드의 호모체를 생산하는 세포주와 제2 폴리펩티드의 호모체를 생산하는 세포주를 혼합하여 배양하고, 당해 배양상청 중에서 FAE 반응을 일으킨 후 정제하는 방법을 들 수 있다. As a non-limiting embodiment of the present invention, a method of culturing each cell line producing a homoform of the first and second polypeptides, respectively, purifying the culture supernatant, and using a purified antibody to cause a FAE (Fab Arm Exchange) reaction; A method of culturing each cell line producing a homoform of the first and second polypeptides, respectively, and mixing the culture supernatant without purification to cause an FAE reaction in the mixed culture supernatant and then purifying, producing a homoform of the first polypeptide A method of generating a FAE reaction using a purified antibody after mixing and culturing a cell line and a cell line producing a homoform of the second polypeptide, and purifying the culture supernatant, a cell line producing a homoform of the first polypeptide and the second polypeptide A method of purifying after mixing and culturing a cell line producing a homo body, causing a FAE reaction in the culture supernatant is mentioned.

본 발명은 비한정의 실시태양으로서, 아래의 a)~c)의 공정；The present invention is a non-limiting embodiment, the steps of a) to c) below;

a) 제1 폴리펩티드의 호모체를 생산하는 세포주와 제2 폴리펩티드의 호모체를 생산하는 세포주를 혼합하는 단계,a) mixing a cell line producing a homo body of the first polypeptide with a cell line producing a homo body of the second polypeptide;

b) 당해 배양상청 중에서 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키도록, 상기 제1 폴리펩티드의 호모체 및 상기 제2 폴리펩티드의 호모체를 함께 인큐베이트하는 단계, 및 b) incubating the homo form of the first polypeptide and the homo form of the second polypeptide together so that cysteine in the hinge region causes isomerization of the disulfide bond in the culture supernatant; and

c) 제1 및 제2 폴리펩티드를 포함하는 이종 다량체를 얻는 단계c) obtaining a heteromultimer comprising the first and second polypeptides;

를 포함하는, 이종 다량체의 제조방법을 제공한다. It provides a method for preparing a heteromultimer comprising a.

본 발명은 비한정의 실시태양으로서, 아래의 a)~c)의 공정；The present invention is a non-limiting embodiment, the steps of a) to c) below;

a) 각각 제1 및 제2 폴리펩티드의 호모체를 생산하는 세포주를 배양하는 단계, a) culturing a cell line that produces homoforms of the first and second polypeptides, respectively;

b) 각 세포주의 배양상청을 혼합하고, 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키도록, 상기 제1 폴리펩티드의 호모체 및 상기 제2 폴리펩티드의 호모체를 함께 인큐베이트하는 단계, 및 b) mixing the culture supernatant of each cell line, and incubating the homoform of the first polypeptide and the homoform of the second polypeptide together so that cysteine in the hinge region causes isomerization of the disulfide bond, and

c) 제1 및 제2 폴리펩티드를 포함하는 이종 다량체를 얻는 단계c) obtaining a heteromultimer comprising the first and second polypeptides;

를 포함하는, 이종 다량체의 제조방법을 제공한다. It provides a method for preparing a heteromultimer comprising a.

목적하는 이종 다량체를 선택하는 방법How to select the desired heteromultimer

또한 본 발명은 목적하는 이종 다량체를 선택하는 방법을 제공한다. 바람직한 태양으로서는, 당해 방법은 아래의 단계를 포함하는 목적하는 특성을 갖는 이종 다량체를 선택하는 방법이다：The present invention also provides a method for selecting a desired heteromultimer. In a preferred embodiment, the method is a method for selecting a heteromultimer having desired properties comprising the steps of:

a) 제1 폴리펩티드의 조 및 제2 폴리펩티드의 조를 제공하는 단계로서, 제1 조를 구성하는 각 폴리펩티드가 제2 조를 구성하는 각 폴리펩티드와 다른 표적 특이성을 가지며, 또한 제1 및 제2 조를 구성하는 각 폴리펩티드가 상기 전하 반발을 이용한 계면 제어에 관한 아미노산 개변 및/또는 CH3영역의 안정성을 불안정하게 하는 아미노산 개변을 포함하는 단계, a) providing a first set of polypeptides and a second set of polypeptides, wherein each polypeptide constituting the first set has a different target specificity than each polypeptide constituting the second set, and wherein each of the first and second sets of polypeptides has a different target specificity Each polypeptide constituting the step comprising amino acid modifications and/or amino acid modifications that destabilize the stability of the CH3 region with respect to the interface control using the charge repulsion,

b) 환원 조건하에서, 상기 제1 조를 구성하는 각 폴리펩티드를 상기 제2 조를 구성하는 각 폴리펩티드와 함께 인큐베이트함으로써 복수 종의 이종 다량체의 혼합물을 제작하는 단계,b) preparing a mixture of a plurality of heteromultimers by incubating each polypeptide constituting the first group with each polypeptide constituting the second group under reducing conditions;

c) 결과로서 얻어진 복수 종의 이종 다량체의 혼합물을 목적하는 특성에 대해서 어세이하는 단계, 및c) assaying the resulting mixture of a plurality of heteromultimers for the desired properties, and

d) 목적하는 특성을 갖는 이종 다량체를 선택하는 단계. d) selecting a heteromultimer with the desired properties.

의약 조성물pharmaceutical composition

또한 본 발명은 본 발명의 이종 다량체 및 의약적으로 허용되는 담체를 포함하는 조성물(약제)에 관한 것이다.The present invention also relates to a composition (pharmaceutical) comprising the heteromultimer of the present invention and a pharmaceutically acceptable carrier.

본 발명에 있어서 의약 조성물이란 통상 질환의 치료 또는 예방, 또는 검사·진단을 위한 약제를 말한다. In the present invention, the pharmaceutical composition usually refers to a drug for treatment or prevention of disease, or examination/diagnosis.

본 발명의 의약 조성물은 당업자에게 공지의 방법으로 제제화하는 것이 가능하다. 예를 들면 물 또는 그 이외의 약학적으로 허용 가능한 액과의 무균성 용액 또는 현탁액제인 주사제의 형태로 비경구적으로 사용할 수 있다. 예를 들면 약리학상 허용되는 담체 또는 매체, 구체적으로는 멸균수나 생리식염수, 식물유, 유화제, 현탁제, 계면활성제, 안정제, 향미제, 부형제, 비히클, 방부제, 결합제 등과 적절히 조합하여, 일반적으로 인정된 제약 실시에 요구되는 단위용량 형태로 혼화함으로써 제제화하는 것을 생각할 수 있다. 이들 제제에 있어서의 유효 성분량은 지시된 범위의 적당한 용량이 얻어지도록 설정한다. The pharmaceutical composition of the present invention can be formulated by a method known to those skilled in the art. For example, it can be used parenterally in the form of an injection, which is a sterile solution or suspension with water or other pharmaceutically acceptable solutions. For example, in appropriate combination with a pharmacologically acceptable carrier or medium, specifically sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., generally recognized It is conceivable to formulate by mixing in the dosage form required for pharmaceutical practice. The amount of active ingredient in these formulations is set so that an appropriate dose within the indicated range is obtained.

주사를 위한 무균 조성물은 주사용 증류수와 같은 비히클을 사용하여 통상의 제제 실시에 따라 처방할 수 있다. The sterile composition for injection can be prescribed according to conventional formulation using a vehicle such as distilled water for injection.

주사용 수용액으로서는, 예를 들면 생리식염수, 포도당이나 기타 보조제(예를 들면 D-소르비톨, D-만나오스, D-만니톨, 염화나트륨)을 포함하는 등장액을 들 수 있다. 적당한 용해 보조제, 예를 들면 알코올(에탄올 등), 폴리알코올(프로필렌글리콜, 폴리에틸렌글리콜 등), 비이온성 계면활성제(폴리소르베이트 80(TM), HCO-50 등)를 병용해도 된다. Examples of the aqueous solution for injection include physiological saline, an isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride). A suitable solubilizing agent, for example, alcohol (ethanol, etc.), polyalcohol (propylene glycol, polyethylene glycol, etc.), and a nonionic surfactant (polysorbate 80(TM), HCO-50, etc.) may be used in combination.

유성액으로서는 참기름, 콩기름을 들 수 있고, 용해 보조제로서 안식향산벤질 및/또는 벤질알코올을 병용해도 된다. 또한 완충제(예를 들면 인산염 완충액 및 초산나트륨 완충액), 무통화제(예를 들면 염산프로카인), 안정제(예를 들면 벤질알코올 및 페놀), 산화방지제와 배합해도 된다. 조제된 주사액은 통상 적당한 앰플에 충전한다. Sesame oil and soybean oil are mentioned as an oily liquid, You may use together benzyl benzoate and/or benzyl alcohol as a solubilizing agent. Moreover, you may mix|blend with a buffer (for example, a phosphate buffer solution and sodium acetate buffer), a soothing agent (for example, procaine hydrochloride), a stabilizer (for example, benzyl alcohol and phenol), and an antioxidant. The prepared injection solution is usually filled in an appropriate ampoule.

본 발명의 의약 조성물은 바람직하게는 비경구 투여에 의해 투여된다. 예를 들면 주사제형, 경비 투여제형, 경폐 투여제형, 경피 투여형의 조성물로 할 수 있다. 예를 들면 정맥내 주사, 근육내 주사, 복강내 주사, 피하 주사 등에 의해 전신 또는 국부적으로 투여할 수 있다. The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, it can be set as the composition of an injection form, a nasal administration form, a transpulmonary administration form, and a transdermal administration form. For example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. can be administered systemically or locally.

투여방법은 환자의 연령, 증상에 따라 적절히 선택할 수 있다. 항체 또는 항체를 코드하는 폴리뉴클레오티드를 함유하는 의약 조성물의 투여량은, 예를 들면 1회에 대해 체중 1 ㎏당 0.0001 ㎎~1,000 ㎎의 범위로 설정하는 것이 가능하다. 또는 예를 들면 환자당 0.001~100,000 ㎎의 투여량으로 하는 것도 가능한데, 본 발명은 반드시 이들 수치에 제한되는 것은 아니다. 투여량 및 투여방법은 환자의 체중, 연령, 증상 등에 따라 변동되는데, 당업자라면 그들 조건을 고려하여 적당한 투여량 및 투여방법을 설정하는 것이 가능하다. The administration method can be appropriately selected according to the age and symptoms of the patient. The dosage of the antibody or the pharmaceutical composition containing the polynucleotide encoding the antibody can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per dose. Alternatively, for example, it is also possible to have a dosage of 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values. The dosage and administration method vary depending on the weight, age, symptoms, etc. of the patient, and those skilled in the art can set the appropriate dosage and administration method in consideration of these conditions.

본 발명에 있어서는 상기 본 발명의 이종 다량체는 암에 대한 치료제 또는 예방제의 유효성분으로서 유용하다. 암은 다음의 것을 포함하는데, 단 이들에만 한정되는 것은 아니다：폐암(소세포폐암, 비소세포폐암, 폐선암 및 폐편평상피암을 포함한다), 대장암, 직장암, 결장암, 유방암, 간암, 위암, 췌장암, 신장암, 전립선암, 난소암, 갑상선암, 담관암, 복막암, 중피종, 편평상피암, 자궁경부암, 자궁체암, 방광암, 식도암, 두경부암, 비인두암, 타액선종양, 흉선종, 피부암, 기저세포종, 악성 흑색종, 항문암, 음경암, 정소암, 빌름스종양, 급성 골수성 백혈병(급성 골수구성 백혈병, 급성 골수아구성 백혈병, 급성 전골수구성 백혈병, 급성 골수단구성 백혈병 및 급성 단구성 백혈병을 포함한다), 만성 골수성 백혈병, 급성 림프성 백혈병, 만성 림프성 백혈병, 호지킨 림프종, 비호지킨 림프종(버킷 림프종, 만성 림프구성 백혈병, 균상 식육종, 맨틀 세포 림프종, 여포성 림프종, 비만성 대세포형 림프종, 변연대 림프종, 모양세포 백혈병 형질세포종, 말초성 T세포 림프종 및 성인 T세포 백혈병/림프종), 랑게르한스 세포조직구증, 다발성 골수종, 골수 이형성 증후군, 뇌종양(신경교종, 성상교종, 교아세포종, 수막종 및 상의 세포종을 포함한다), 신경아세포종, 망막아세포종, 골육종, 카포시 육종, 유윙 육종, 혈관육종, 혈관외피세포종.In the present invention, the heteromultimer of the present invention is useful as an active ingredient in a therapeutic or prophylactic agent for cancer. Cancers include, but are not limited to: lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cancer), colorectal cancer, rectal cancer, colon cancer, breast cancer, liver cancer, stomach cancer, pancreatic cancer , kidney cancer, prostate cancer, ovarian cancer, thyroid cancer, cholangiocarcinoma, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, uterine body cancer, bladder cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, salivary gland tumor, thymoma, skin cancer, basal cell carcinoma, malignant black tumor, anal cancer, penile cancer, testicular cancer, Wilms' tumor, acute myeloid leukemia (including acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia and acute monocytic leukemia) , chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Buckett's lymphoma, chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, obese large cell lymphoma, Marginal zone lymphoma, cystic cell leukemia plasmacytoma, peripheral T-cell lymphoma and adult T-cell leukemia/lymphoma), Langerhans cell histiocytosis, multiple myeloma, myelodysplastic syndrome, brain tumors (glioma, astroglioma, glioblastoma, meningioma and ependymoma) ), neuroblastoma, retinoblastoma, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma, hemangiosarcoma, hemangioepitheloma.

또한 필요에 따라 본 발명의 폴리펩티드 또는 이종 다량체를 그 밖의 의약 성분과 조합하여 제제화하는 것도 가능하다. It is also possible to formulate the polypeptide or heteromultimer of the present invention in combination with other pharmaceutical ingredients, if necessary.

또한 본 발명은 적어도 본 발명의 제조방법에 의해 제조된 이종 다량체, 또는 본 발명의 의약 조성물을 포함하는 본 발명의 치료방법 또는 예방방법에 사용하기 위한 키트를 제공한다. 상기 키트에는 그 밖에 약학적으로 허용되는 담체, 매체, 사용방법을 기재한 지시서 등을 팩키징해두는 것도 가능하다. 또한 본 발명은 본 발명의 폴리펩티드 또는 본 발명의 제조방법에 의해 제조된 폴리펩티드의 면역 염증성 질환의 치료제 또는 예방제의 제조에 있어서의 사용에 관한 것이다. 또한 본 발명은 본 발명의 치료방법 또는 예방방법에 사용하기 위한 본 발명의 폴리펩티드 또는 본 발명의 제조방법에 의해 제조된 폴리펩티드에 관한 것이다. The present invention also provides a kit for use in the treatment or prevention method of the present invention, comprising at least the heteromultimer produced by the method of the present invention, or the pharmaceutical composition of the present invention. In the kit, other pharmaceutically acceptable carriers, media, instructions for use, etc. may be packaged. The present invention also relates to the use of the polypeptide of the present invention or the polypeptide produced by the method of the present invention in the manufacture of an agent for the treatment or prophylaxis of immune inflammatory diseases. The present invention also relates to a polypeptide of the present invention for use in a method of treatment or prevention of the present invention or a polypeptide produced by the method of the present invention.

또한 본 명세서에서 사용되고 있는 아미노산의 3문자 표기와 1문자 표기의 대응은 아래와 같다. In addition, the correspondence between the three-letter notation and the one-letter notation of amino acids used in the present specification is as follows.

알라닌：Ala：AAlanine: Ala: A

아르기닌：Arg：RArginine: Arg: R

아스파라긴：Asn：NAsparagine: Asn: N

아스파라긴산：Asp：DAspartic acid: Asp: D

시스테인：Cys：CCysteine: Cys: C

글루타민：Gln：QGlutamine: Gln: Q

글루타민산：Glu：EGlutamic acid: Glu: E

글리신：Gly：GGlycine: Gly: G

히스티딘：His：HHistidine: His: H

이소류신：Ile：IIsoleucine: Ile: I

류신：Leu：LLeu: Leu: L

리신：Lys：KLysine: Lys: K

메티오닌：Met：MMethionine: Met: M

페닐알라닌：Phe：FPhenylalanine: Phe: F

프롤린：Pro：PProline: Pro: P

세린：Ser：SSerine: Ser: S

트레오닌：Thr：TThreonine: Thr: T

트립토판：Trp：WTryptophan: Trp: W

티로신：Tyr：YTyrosine: Tyr: Y

발린：Val：VValine: Val: V

또한 본 명세서에 있어서 인용된 모든 선행기술문헌은 참조로써 본 명세서에 포함된다. Also, all prior art documents cited in this specification are incorporated herein by reference.

실시예Example

〔실시예 1〕항체의 회합 계면 제어 개변의 도입에 의한 Fab Arm Exchange 효율의 향상 검토[Example 1] Study on improvement of Fab arm exchange efficiency by introduction of antibody association interface control modification

Fab Arm Exchange의 경우는, 2종류의 호모항체를 환원제 존재하에서 혼합하고, 생성된 4개의 항체 분자 편완(반분자 또는 HL분자라 칭하는, 1개의 중쇄 및 1개의 경쇄로 이루어지는 분자)이 재결합됨으로써 이중특이성 항체(Bispecific antibody)가 생성된다. HL분자의 재결합은 랜덤으로 일어나기 때문에 목적의 이중특이성 항체는 이론상 시스템 중에 존재하는 전체 항체량의 50%밖에 얻어지지 않는다. 그러나 2종류의 호모항체에 사전에 다른 전하를 도입해 둠으로써, 생성된 HL분자끼리가 재결합될 때 호모 이량화보다도 헤테로 이량화가 보다 우선적으로 일어나 높은 효율로 이중특이성 항체를 제작할 수 있을 가능성이 있다. 이에 WO2006/106905에서 보고되어 있는 항체의 CH3영역에 있어서의 회합 계면을 제어하는 개변(2종류의 H쇄를 CH3영역의 전하적 상호작용 및 반발을 이용하여 헤테로 회합화시키는 개변)을 사용함으로써 Fab Arm Exchange의 반응 효율(이중특이성 항체 생성률)이 향상되는지 여부를 검증하였다. In the case of Fab Arm Exchange, two types of homoantibodies are mixed in the presence of a reducing agent, and the resulting four antibody molecule fragments (referred to as semimolecules or HL molecules, molecules consisting of one heavy chain and one light chain) are recombined to double A bispecific antibody is generated. Since recombination of HL molecules occurs randomly, the target bispecific antibody is theoretically obtained only 50% of the total amount of antibody present in the system. However, by introducing different charges to the two types of homoantibodies in advance, when the generated HL molecules are reunited with each other, heterodimerization takes precedence over homodimerization, and there is a possibility that a bispecific antibody can be produced with high efficiency. . Therefore, by using the modification to control the association interface in the CH3 region of the antibody reported in WO2006/106905 (modification to heteroassociate two types of H chains using the charge interaction and repulsion of the CH3 region), Fab Arm It was verified whether the reaction efficiency (bispecific antibody production rate) of Exchange was improved.

항체 H쇄 가변영역에는 WO2009/125825에 개시되어 있는 인간 인터류킨 6 수용체에 대한 항체의 H쇄 가변영역인 WT(H)(서열번호 1：이후 MRAH로 기재) 및 H54(서열번호 2)를 사용하였다. 이들 H쇄 가변영역에 대해 항체 H쇄 불변영역으로서 인간 IgG1의 H쇄 불변영역의 C말단의 Gly 및 Lys를 제거한 G1d를 갖는 MRAH-G1d(서열번호 3) 및 H54-G1d(서열번호 4), 및 인간 IgG4의 H쇄 불변영역의 C말단의 Gly 및 Lys를 제거한 wtG4d를 갖는 MRAH-wtG4d(서열번호 5) 및 H54-wtG4d(서열번호 6)를 제작하였다. 다음으로 MRAH-G1d와 H54-G1d에 대해 IgG4형 힌지 서열, CH3 도메인 서열이 되도록 P228S 및 K409R의 개변을 도입한 MRAH-G1dsr(서열번호 7), H54-G1dsr(서열번호 8)을 제작하였다. 또한 회합 계면 제어 개변으로서 MRAH-G1dsr에 D356K를 도입한 MRAH-G1dsrP1(서열번호 9), H54-G1dsr에 K439E를 도입한 H54-G1dsrN1(서열번호 10), MRAH-wtG4d에 E356K를 도입한 MRAH-wtG4dP1(서열번호 11), H54-wtG4d에 K439E를 도입한 H54-wtG4dN1(서열번호 12)을 제작하였다. H쇄 가변영역이 MRAH인 경우는 MRAL-k0(서열번호 13)를, H54인 경우는 L28-k0(서열번호 14)를 항체 L쇄로서 사용하였다. 참고예 1의 방법에 따라 MRAH-G1dsr/MRAL-k0, H54-G1dsr/L28-k0, MRAH-G1dsrP1/MRAL-k0, H54-G1dsrN1/L28-k0, MRAH-wtG4d/MRAL-k0, H54-wtG4d/L28-k0, MRAH-wtG4dP1/MRAL-k0, H54-wtG4dN1/L28-k0를 발현, 정제하였다. For the antibody H chain variable region, WT (H) (SEQ ID NO: 1: hereinafter referred to as MRAH) and H54 (SEQ ID NO: 2), which are the H chain variable regions of the antibody against human interleukin 6 receptor disclosed in WO2009/125825 were used. . For these H chain variable regions, MRAH-G1d (SEQ ID NO: 3) and H54-G1d (SEQ ID NO: 4) having G1d from which Gly and Lys at the C-terminus of the H chain constant region of human IgG1 are removed as antibody H chain constant regions; and MRAH-wtG4d (SEQ ID NO: 5) and H54-wtG4d (SEQ ID NO: 6) having wtG4d in which Gly and Lys at the C-terminus of the H chain constant region of human IgG4 were removed. Next, for MRAH-G1d and H54-G1d, MRAH-G1dsr (SEQ ID NO: 7) and H54-G1dsr (SEQ ID NO: 8) were prepared in which modifications of P228S and K409R were introduced to become an IgG4-type hinge sequence and a CH3 domain sequence. In addition, as an association interface control modification, MRAH-G1dsrP1 (SEQ ID NO: 9) introduced with D356K into MRAH-G1dsr, H54-G1dsrN1 with K439E introduced into H54-G1dsr (SEQ ID NO: 10), MRAH- with E356K introduced into MRAH-wtG4d H54-wtG4dN1 (SEQ ID NO: 12) in which K439E was introduced into wtG4dP1 (SEQ ID NO: 11) and H54-wtG4d was prepared. When the H chain variable region was MRAH, MRAL-k0 (SEQ ID NO: 13) was used, and in the case of H54, L28-k0 (SEQ ID NO: 14) was used as the antibody L chain. According to the method of Reference Example 1, MRAH-G1dsr/MRAL-k0, H54-G1dsr/L28-k0, MRAH-G1dsrP1/MRAL-k0, H54-G1dsrN1/L28-k0, MRAH-wtG4d/MRAL-k0, H54-wtG4d /L28-k0, MRAH-wtG4dP1/MRAL-k0, H54-wtG4dN1/L28-k0 were expressed and purified.

다음으로 얻어진 호모체 2종류를 아래에 나타내는 조합으로 혼합하고, 반응산물을 참고예 2의 방법에 따라 평가하였다. Next, two types of the obtained homo bodies were mixed in the combination shown below, and the reaction product was evaluated according to the method of Reference Example 2.

(1) MRAH-wtG4d/MRAL-k0와 H54-wtG4d/L28-k0(1) MRAH-wtG4d/MRAL-k0 and H54-wtG4d/L28-k0

(2) MRAH-wtG4dP1/MRAL-k0와 H54-wtG4dN1/L28-k0(2) MRAH-wtG4dP1/MRAL-k0 and H54-wtG4dN1/L28-k0

(3) MRAH-G1dsr/MRAL-k0와 H54-G1dsr/L28-k0(3) MRAH-G1dsr/MRAL-k0 and H54-G1dsr/L28-k0

(4) MRAH-G1dsrP1/MRAL-k0와 H54-G1dsrN1/L28-k0(4) MRAH-G1dsrP1/MRAL-k0 and H54-G1dsrN1/L28-k0

반응 조건：PBS(Sigma, pH 7.4) 중 [각 mAb] = 0.2 ㎎/㎖, [GSH(Sigma)] = 0.5 mM, 0.05% Tween 20 (Junsei), 37℃, 24시간. Reaction conditions: [each mAb] = 0.2 mg/ml, [GSH (Sigma)] = 0.5 mM, 0.05% Tween 20 (Junsei) in PBS (Sigma, pH 7.4), 37°C, 24 hours.

본 검토에서 사용한 2종류의 항체 가변영역 MRAH/MRAL과 H54/L28은 pI가 크게 상이하기 때문에, 이온 교환 크로마토그래피에 의해 각각의 호모체 및 생성된 이중특이성 항체에 대응하는 피크가 용이하게 분리되어 반응 효율을 평가할 수 있다. 도 1은 이온 교환 크로마토그래피에 의해 반응산물을 평가한 결과이다. 회합 계면 제어 개변을 도입하지 않는 MRAH-wtG4d/MRAL-k0와 H54-wtG4d/L28-k0를 반응시킨 wtG4d, 또는 MRAH-G1dsr/MRAL-k0와 H54-G1dsr/L28-k0를 반응시킨 G1dsr의 이중특이성 항체 생성률은 각각 50.5%, 52.7%였다. 한편으로 회합 계면 제어 개변을 도입한 MRAH-wtG4dP1/MRAL-k0와 H54-wtG4dN1/L28-k0를 반응시킨 wtG4dP1/N1의 이중특이성 항체 생성률은 99.0%, 또한 MRAH-G1dsrP1/MRAL-k0와 H54-G1dsrN1/L28-k0를 반응시킨 G1dsrP1/N1의 경우는 98.5%로 매우 높은 효율로 이중특이성 항체가 생성되고 있는 것이 명확해졌다. 이상의 결과로부터, WO2006/106905에서 보고되어 있는 회합 계면 제어 개변을 도입한 2종류의 호모체를 환원제 존재하에서 혼합함으로써 매우 높은 효율로 이중특이성 항체를 제작할 수 있는 것이 나타내어졌다. Since the two types of antibody variable regions MRAH/MRAL and H54/L28 used in this study have significantly different pIs, the peaks corresponding to each homoform and the generated bispecific antibody are easily separated by ion exchange chromatography. The reaction efficiency can be evaluated. 1 is a result of evaluating the reaction product by ion exchange chromatography. The double of MRAH-wtG4d/MRAL-k0 and H54-wtG4d/L28-k0, which did not introduce the associative interface control modification, or G1dsr, which reacted with MRAH-G1dsr/MRAL-k0 and H54-G1dsr/L28-k0 The specific antibody production rates were 50.5% and 52.7%, respectively. On the other hand, the bispecific antibody production rate of wtG4dP1/N1 reacted with MRAH-wtG4dP1/MRAL-k0 and H54-wtG4dN1/L28-k0 introduced with association interface control modifications was 99.0%, and MRAH-G1dsrP1/MRAL-k0 and H54- In the case of G1dsrP1/N1 reacted with G1dsrN1/L28-k0, it became clear that the bispecific antibody was being generated with a very high efficiency (98.5%). From the above results, it was shown that bispecific antibodies can be produced with very high efficiency by mixing two types of homoforms introduced with the association interface control modification reported in WO2006/106905 in the presence of a reducing agent.

〔실시예 2〕인간 천연형 IgG1의 힌지 서열을 갖는 호모체에서의 Fab Arm Exchange[Example 2] Fab Arm Exchange in a Homobody Having a Hinge Sequence of Human Native IgG1

실시예 1에서는 힌지영역의 서열을 천연형 인간 IgG4형으로 하기 위해 IgG1에 대해 P228S 개변을 도입하여 Fab-Arm Exchange를 행하였다. 그러나 생체 내에 투여된 천연형 IgG4는 내인성 IgG4와의 반분자 교환이 일어나는 것이 보고되어 있다. 또한 그 원인은 힌지영역 중 EU 넘버링 228번째에 위치하는 Ser로, 이를 IgG1형의 Pro로 함으로써 안정성이 향상되고, 생체 내에서의 교환이 일어나지 않게 되는 것이 보고되어 있다(Labrijn AF et al. Nat. Biotechnol. 2009, 27, 767-771). 따라서 생체 내에 투여하는 것을 고려한 경우, 제작한 이중특이성 항체의 힌지 서열은 226C-227P-228P-229C로 되어 있는 것이 바람직하다. 이에 본 검토에서는 회합 계면 제어 개변이 도입되어 있는 것으로 인해 천연형 인간 IgG1의 힌지 서열이더라도 효율적으로 Fab Arm Exchange가 일어나는지 여부에 대해서 검증하였다.In Example 1, Fab-Arm Exchange was performed by introducing P228S modification to IgG1 in order to change the hinge region sequence to a native human IgG4 type. However, it has been reported that half-molecular exchange with endogenous IgG4 occurs in native IgG4 administered in vivo. In addition, it has been reported that the cause is Ser located at the 228th EU numbering position in the hinge region, and by making it an IgG1 type Pro, stability is improved, and exchange in vivo does not occur (Labrijn AF et al. Nat. Biotechnol. 2009, 27, 767-771). Therefore, in consideration of in vivo administration, the hinge sequence of the prepared bispecific antibody is preferably 226C-227P-228P-229C. Therefore, in this review, it was verified whether Fab arm exchange occurs efficiently even with the hinge sequence of native human IgG1 due to the introduction of the associative interface control modification.

먼저 MRAH-G1d에 대해 K409R 및 D356K를 도입한 MRAH-G1drP1(서열번호 15), H54-G1d에 대해 K409R 및 K439E를 도입한 H54-G1drN1(서열번호 16)을 제작하였다. H쇄 가변영역이 MRAH인 경우는 MRAL-k0를, H54인 경우는 L28-k0를 항체 L쇄로서 사용하고, 참고예 1의 방법에 따라 MRAH-G1drP1/MRAL-k0, H54-G1drN1/L28-k0를 발현, 정제하였다. 다음으로, 얻어진 호모체 2종류를 아래의 반응 조건 하에서 혼합하고, 반응산물을 참고예 2의 방법에 따라 평가하였다. First, MRAH-G1drP1 (SEQ ID NO: 15) introduced with K409R and D356K for MRAH-G1d, and H54-G1drN1 (SEQ ID NO: 16) introduced with K409R and K439E for H54-G1d were prepared. When the H chain variable region is MRAH, MRAL-k0 is used, and when it is H54, L28-k0 is used as the antibody L chain, and according to the method of Reference Example 1, MRAH-G1drP1/MRAL-k0, H54-G1drN1/L28- k0 was expressed and purified. Next, two types of the obtained homo bodies were mixed under the following reaction conditions, and the reaction product was evaluated according to the method of Reference Example 2.

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 0.2 ㎎/㎖, 0.05% Tween 20 (Junsei), 37℃, 24시간. 환원제는 [GSH(Sigma)] = 0.5 mM 또는 5 mM 또는 [2-MEA(Sigma)] = 25 mM의 3 조건에서 검토하였다. Reaction conditions: [each mAb] in TBS (Takara, pH 7.6) = 0.2 mg/ml, 0.05% Tween 20 (Junsei), 37°C, 24 hours. The reducing agent was studied under 3 conditions of [GSH(Sigma)] = 0.5 mM or 5 mM or [2-MEA(Sigma)] = 25 mM.

참고예 2의 방법에 따라 반응산물을 해석한 결과를 도 2에 나타낸다. 실시예 1과 동일한 GSH(0.5 mM)의 조건하에서는 이중특이성 항체 생성률은 21.8%가 되어, EU 넘버링 228번째 아미노산 잔기가 Ser인 경우와 비교하여 효율이 대폭 저하되었다. 그러나 2-MEA(25 mM) 및 GSH(5 mM)의 환원 조건하에서는 이중특이성 항체의 생성률이 99% 이상이 되었다. 이상의 결과로부터, 힌지의 서열이 인간 천연형 IgG1의 서열이었다 하더라도, 회합 계면 제어 개변을 도입하고 적절한 환원 조건을 사용함으로써 높은 효율로 이중특이성 항체를 제작할 수 있는 것이 명확해졌다. The result of analyzing the reaction product according to the method of Reference Example 2 is shown in FIG. 2 . Under the same conditions of GSH (0.5 mM) as in Example 1, the bispecific antibody production rate was 21.8%, and the efficiency was significantly reduced compared to the case where the 228th amino acid residue EU numbering was Ser. However, under the reducing conditions of 2-MEA (25 mM) and GSH (5 mM), the production rate of the bispecific antibody was more than 99%. From the above results, it was clarified that even if the hinge sequence was a sequence of natural human IgG1, a bispecific antibody could be produced with high efficiency by introducing association interface control modifications and using appropriate reducing conditions.

〔실시예 3〕인간 천연형 IgG1의 CH3를 사용한 Fab Arm Exchange[Example 3] Fab Arm Exchange Using CH3 of Natural Human IgG1

여기까지의 검토에 있어서 인간 IgG1에 대해 CH3를 IgG4형으로 하는 K409R 개변과, 회합 계면 제어 개변(D356K 및 K439E)을 도입하여 Fab Arm Exchange를 행함으로써 매우 높은 효율로 목적의 이중특이성 항체가 얻어지는 것이 나타내어졌다. In the studies up to this point, by introducing K409R modification in which CH3 is IgG4 type to human IgG1 and Fab arm exchange by introducing association interface control modification (D356K and K439E), the target bispecific antibody can be obtained with very high efficiency. was shown.

한편 409번째 아미노산 잔기가 Arg이면 항체의 산성 조건하에 있어서의 안정성이 저하되는 것이 알려져 있다(WO/2009/041613). 의약품으로서의 항체의 제조를 위해서는 산성 조건하에 항체를 폭로하는 바이러스 불활성화 공정이 필수이고, 그때 항체의 품질을 유지하기 위해 항체의 산성 조건하에 있어서의 안정성이 높은 것이 바람직하다. 따라서 409번째 아미노산 잔기는 Arg가 아닌 것이 바람직하다. 한편 Fab Arm Exchange 반응을 효율적으로 일으킬 수 있는 것으로 보고되어 있는 개변은 K409R의 개변을 사용하고 있어, 409번째 아미노산 잔기가 Arg인 것으로부터 산성 조건하에 있어서의 안정성에 과제가 있는 것으로 생각된다. 이에 다음으로 K409R 개변을 포함하지 않는 완전히 인간 천연형 IgG1의 항체에 대해 WO2006/106905에서 보고되어 있는 회합 계면 제어 개변만으로 Fab Arm Exchange가 야기되는지 여부를 검증하였다. On the other hand, when the 409th amino acid residue is Arg, it is known that the stability of the antibody under acidic conditions is reduced (WO/2009/041613). In order to manufacture an antibody as a pharmaceutical, a virus inactivation step in which the antibody is exposed under acidic conditions is essential, and in that case, in order to maintain the quality of the antibody, it is preferable that the antibody has high stability under acidic conditions. Therefore, it is preferable that the 409th amino acid residue is not Arg. On the other hand, the modification reported to be able to efficiently cause the Fab arm exchange reaction uses the modification of K409R, and since the 409th amino acid residue is Arg, it is considered that there is a problem in stability under acidic conditions. Next, it was verified whether Fab Arm Exchange was caused only by the association interface control modification reported in WO2006/106905 for a completely human native IgG1 antibody that does not include the K409R modification.

검토한 회합 계면 제어 개변의 조합을 표 1에 나타낸다. Table 1 shows the combinations of the examined association interface control modifications.

Figure 112016038698342-pct00001
Figure 112016038698342-pct00001

H쇄 가변영역이 MRAH인 경우는 MRAL-k0를, H54인 경우는 L28-k0를 항체 L쇄로서 사용하였다. 참고예 1의 방법에 따라 MRAH-G1dP1/MRAL-k0, H54-G1dN1/L28-k0, MRAH-G1dP3/MRAL-k0, H54-G1dN3/L28-k0, MRAH-G1dP4/MRAL-k0, H54-G1dN4/L28-k0, MRAH-G1dP5/MRAL-k0, H54-G1dN5/L28-k0, MRAH-G1dP6/MRAL-k0, H54-G1dN6/L28-k0를 발현, 정제하였다.When the H chain variable region was MRAH, MRAL-k0 was used, and in the case of H54, L28-k0 was used as the antibody L chain. According to the method of Reference Example 1, MRAH-G1dP1/MRAL-k0, H54-G1dN1/L28-k0, MRAH-G1dP3/MRAL-k0, H54-G1dN3/L28-k0, MRAH-G1dP4/MRAL-k0, H54-G1dN4 /L28-k0, MRAH-G1dP5/MRAL-k0, H54-G1dN5/L28-k0, MRAH-G1dP6/MRAL-k0, H54-G1dN6/L28-k0 were expressed and purified.

다음으로 얻어진 호모체 2종류를 아래에 나타내는 조합으로 혼합하고, 반응산물을 참고예 2의 방법에 따라 평가하였다. Next, two types of the obtained homo bodies were mixed in the combination shown below, and the reaction product was evaluated according to the method of Reference Example 2.

(1) MRAH-G1dP1/MRAL-k0와 H54-G1dN1/L28-k0(1) MRAH-G1dP1/MRAL-k0 and H54-G1dN1/L28-k0

(2) MRAH-G1dP3/MRAL-k0와 H54-G1dN3/L28-k0(2) MRAH-G1dP3/MRAL-k0 and H54-G1dN3/L28-k0

(3) MRAH-G1dP4/MRAL-k0와 H54-G1dN4/L28-k0(3) MRAH-G1dP4/MRAL-k0 and H54-G1dN4/L28-k0

(4) MRAH-G1dP5/MRAL-k0와 H54-G1dN5/L28-k0(4) MRAH-G1dP5/MRAL-k0 and H54-G1dN5/L28-k0

(5) MRAH-G1dP6/MRAL-k0와 H54-G1dN6/L28-k0(5) MRAH-G1dP6/MRAL-k0 and H54-G1dN6/L28-k0

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 0.2 ㎎/㎖, 0.05% Tween 20 (Junsei), [GSH(Sigma)] = 5 mM, 37℃, 24시간. Reaction conditions: [each mAb] = 0.2 mg/ml, 0.05% Tween 20 (Junsei), [GSH (Sigma)] = 5 mM, 37 °C, 24 hours in TBS (Takara, pH 7.6).

얻어진 결과를 표 2에 나타낸다.The obtained results are shown in Table 2.

Figure 112016038698342-pct00002
Figure 112016038698342-pct00002

표 중의 「약칭」이란, 반응에 사용한 호모체의 조합의 약칭을 나타내는 것이다. 예를 들면 약칭이 G1dP1/N1으로 되어 있었던 경우, MRAH-G1dP1/MRAL-k0와 H54-G1dN1/L28-k0를 반응시킨 것을 나타낸다. 또한 「사용한 MoAb1의 H쇄 불변영역명」이란, MRAH의 가변영역을 갖는 항체의 불변영역명을 나타낸 것이고, 「사용한 MoAb2의 H쇄 불변영역명」이란, H54의 가변영역을 갖는 항체의 불변영역명을 나타낸 것이다. 「도입한 개변」이란, MRAH-G1d 또는 H54-G1d에 대해 도입한 개변을 나타낸다. "Abbreviation" in a table|surface shows the abbreviation of the combination of the homo body used for reaction. For example, when the abbreviation is G1dP1/N1, it indicates that MRAH-G1dP1/MRAL-k0 and H54-G1dN1/L28-k0 are reacted. In addition, "the name of the H chain constant region of MoAb1 used" indicates the constant region name of an antibody having a variable region of MRAH, and "the name of the H chain constant region of MoAb2 used" is a constant region of an antibody having a variable region of H54. name is indicated. "Introduced modification" refers to a modification introduced with respect to MRAH-G1d or H54-G1d.

한쪽 호모체에 D356K를, 다른 한쪽 호모체에 K439E를 도입한 G1dP1/N1에서는 이중특이성 항체 생성률이 1.7%였다. 도 2에 있어서 같은 반응 조건하(5 mM GSH)에서 K409R 개변과 회합 계면 제어 개변(D356K 및 K439E)을 갖는 G1drP1/N1의 이중특이성 항체 생성률은 99.3%였던 것에 반하여, EU 넘버링 409번째 아미노산 잔기가 Lys가 된 G1dP1/N1에서 반응 효율이 대폭 저하되는 것이 나타내어졌다. 그러나 한쪽 호모체에 회합 계면 제어 개변 D399K를, 다른 한쪽 호모체에 K409D를 도입한 G1dP3/N3, 한쪽 호모체에 D356K/D399K를, 다른 한쪽 호모체에 K409D/K439E를 도입한 G1dP5/N5는 각각 93.4%, 98.1%로 매우 높은 이중특이성 항체 생성률을 나타내었다. 따라서 CH3 도메인을 IgG4형으로 하는 K409R 개변을 사용하지 않고 회합 계면 제어 개변만으로 높은 효율로 Fab Arm Exchange를 야기할 수 있는 것이 나타내어졌다. In G1dP1/N1, in which D356K was introduced into one homo body and K439E was introduced into the other homo body, the bispecific antibody production rate was 1.7%. In Figure 2, the bispecific antibody production rate of G1drP1/N1 having K409R modification and association interface control modification (D356K and K439E) under the same reaction conditions (5 mM GSH) was 99.3%, whereas the EU numbering 409th amino acid residue was It was shown that the reaction efficiency was significantly reduced in G1dP1/N1 as Lys. However, G1dP3/N3 introduced with the association interface control modification D399K into one homo body, K409D K409D into the other homo body, D356K/D399K into one homo body, and G1dP5/N5 introduced with K409D/K439E into the other homo body, respectively. They showed very high bispecific antibody production rates of 93.4% and 98.1%. Therefore, it was shown that Fab Arm Exchange can be caused with high efficiency only by the association interface control modification without using the K409R modification having the CH3 domain as an IgG4 type.

다음으로 반응 효율이 높았던 G1dP3/N3와 G1dP5/N5에 대해서 3종류의 환원 조건하에서 반응 효율을 비교하였다. 이때 비교 대상으로서 실시예 2에서 사용한 G1drP1/N1 및 Fab Arm Exchange에 의한 효율적인 이중특이성 항체 제작 개변으로서 Labrijn 등에 의해 보고되어 있는, 한쪽 항체에 K409R을 다른 한쪽 호모체에 F405L을 도입한 개변체의 조합에 대해서도 검증하였다(Labrijn AF et al. Proc. Natl., Acad. Sci., 2013. 110. 5145-5150). Next, the reaction efficiency of G1dP3/N3 and G1dP5/N5, which had high reaction efficiency, was compared under three types of reducing conditions. At this time, as a comparison object, G1drP1/N1 and Fab Arm Exchange used in Example 2 as an efficient bispecific antibody production modification by Labrijn et al. A combination of variants introduced by K409R in one antibody and F405L in the other homo body as reported by Labrijn et al. was also verified (Labrijn AF et al. Proc. Natl., Acad. Sci., 2013. 110. 5145-5150).

MRAH-G1d에 대해 K409R을 도입한 MRAH-G1dr(서열번호 27), H54-G1d에 대해 F405L을 도입한 H54-G1dl(서열번호 28)을 제작하였다. H쇄 가변영역이 MRAH인 경우는 MRAL-k0를, H54인 경우는 L28-k0를 항체 L쇄로서 사용하였다. 참고예 1의 방법에 따라 MRAH-G1drP1/MRAL-k0, H54-G1drN1/L28-k0, MRAH-G1dP3/MRAL-k0, H54-G1dN3/L28-k0, MRAH-G1dP5/MRAL-k0, H54-G1dN5/L28-k0, MRAH-G1dr/MRAL-k0, H54-G1dl/L28-k0를 발현, 정제하였다.MRAH-G1dr (SEQ ID NO: 27) introduced with K409R for MRAH-G1d and H54-G1dl (SEQ ID NO: 28) introduced with F405L for H54-G1d were prepared. When the H chain variable region was MRAH, MRAL-k0 was used, and in the case of H54, L28-k0 was used as the antibody L chain. According to the method of Reference Example 1, MRAH-G1drP1/MRAL-k0, H54-G1drN1/L28-k0, MRAH-G1dP3/MRAL-k0, H54-G1dN3/L28-k0, MRAH-G1dP5/MRAL-k0, H54-G1dN5 /L28-k0, MRAH-G1dr/MRAL-k0, H54-G1dl/L28-k0 were expressed and purified.

다음으로 얻어진 호모체 2종류를 아래에 나타내는 조합으로 혼합하고, 반응산물을 참고예 2의 방법에 따라 평가하였다. Next, two types of the obtained homo bodies were mixed in the combination shown below, and the reaction product was evaluated according to the method of Reference Example 2.

(1) MRAH-G1drP1/MRAL-k0와 H54-G1drN1/L28-k0(1) MRAH-G1drP1/MRAL-k0 and H54-G1drN1/L28-k0

(2) MRAH-G1dP3/MRAL-k0와 H54-G1dN3/L28-k0(2) MRAH-G1dP3/MRAL-k0 and H54-G1dN3/L28-k0

(3) MRAH-G1dP5/MRAL-k0와 H54-G1dN5/L28-k0(3) MRAH-G1dP5/MRAL-k0 and H54-G1dN5/L28-k0

(4) MRAH-G1dr/MRAL-k0와 H54-G1dl/L28-k0(4) MRAH-G1dr/MRAL-k0 and H54-G1dl/L28-k0

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 0.2 ㎎/㎖, 0.05% Tween 20 (Junsei), 37℃, 24시간. 환원제는 [GSH(Sigma)] = 0.5 mM 또는 5 mM 또는 [2-MEA(Sigma)] = 25 mM의 3 조건에서 검토하였다. Reaction conditions: [each mAb] in TBS (Takara, pH 7.6) = 0.2 mg/ml, 0.05% Tween 20 (Junsei), 37°C, 24 hours. The reducing agent was studied under 3 conditions of [GSH(Sigma)] = 0.5 mM or 5 mM or [2-MEA(Sigma)] = 25 mM.

얻어진 결과를 표 3에 나타낸다.The obtained results are shown in Table 3.

Figure 112016038698342-pct00003
Figure 112016038698342-pct00003

표 중의 「약칭」이란, 반응에 사용한 호모체의 조합의 약칭을 나타내는 것이다. 예를 들면 약칭이 G1dP1/N1으로 되어 있었던 경우, MRAH-G1dP1/MRAL-k0와 H54-G1dN1/L28-k0를 반응시킨 것을 나타낸다. 또한 「사용한 MoAb1의 H쇄 불변영역명」이란, MRAH의 가변영역을 갖는 항체의 불변영역명을 나타낸 것이고, 「사용한 MoAb2의 H쇄 불변영역명」이란, H54의 가변영역을 갖는 항체의 불변영역명을 나타낸 것이다. 「도입한 개변」이란, MRAH-G1d 또는 H54-G1d에 대해 도입한 개변을 나타낸다. "Abbreviation" in a table|surface shows the abbreviation of the combination of the homo body used for reaction. For example, when the abbreviation is G1dP1/N1, it indicates that MRAH-G1dP1/MRAL-k0 and H54-G1dN1/L28-k0 are reacted. In addition, "the name of the H chain constant region of MoAb1 used" indicates the constant region name of an antibody having a variable region of MRAH, and "the name of the H chain constant region of MoAb2 used" is a constant region of an antibody having a variable region of H54. name is indicated. "Introduced modification" refers to a modification introduced with respect to MRAH-G1d or H54-G1d.

5 mM GSH의 환원 조건하에서는 Labrijn 등에 의해 보고되어 있는 기존의 Fab Arm Exchange 효율 향상 개변을 사용한 G1dr/l의 이중특이성 항체 생성률은 87.3%였다. 이때 한쪽 호모체에 D399K를, 다른 한쪽 호모체에 K409D를 도입한 G1dP3/N3의 경우는 85.2%, 한쪽 호모체에 D356K/D399K를, 다른 한쪽 호모체에 K409D/K439E를 도입한 G1dP5/N5의 경우는 99.1%였다. 또한 IgG4형의 K409R 개변에 더하여 한쪽 호모체에 D356K를 다른 한쪽 호모체에 K439E를 갖는 G1drP1/N1의 이중특이성 항체 생성률은 99.3%였다.Under the reducing conditions of 5 mM GSH, the production rate of the bispecific antibody of G1dr/l using the existing Fab arm exchange efficiency improvement modification reported by Labrijn et al. was 87.3%. At this time, in the case of G1dP3/N3 in which D399K was introduced into one homo body and K409D was introduced in the other homo body, 85.2%, D356K/D399K in one homo body, and K409D/K439E in the other homo body were introduced in G1dP5/N5. case was 99.1%. In addition, the production rate of the bispecific antibody of G1drP1/N1 having D356K in one homo body and K439E in the other homo body in addition to the K409R modification of the IgG4 type was 99.3%.

25 mM 2MEA의 환원 조건하에서는 Labrijn 등에 의해 보고되어 있는 기존의 Fab Arm Exchange 효율 향상 개변을 사용한 G1dr/l의 이중특이성 항체 생성률은 95.6%였다. 이때 한쪽 호모체에 D399K를, 다른 한쪽 호모체에 K409D를 도입한 G1dP3/N3의 경우는 92.8%, 한쪽 호모체에 D356K/D399K를 다른 한쪽 호모체에 K409D/K439E를 도입한 G1dP5/N5의 경우는 100%, 또한 IgG4형의 K409R 개변에 더하여 한쪽 호모체에 D356K를 다른 한쪽 호모체에 K439E를 갖는 G1drP1/N1의 이중특이성 항체 생성률은 99.7%였다. Under the reducing conditions of 25 mM 2MEA, the production rate of the bispecific antibody of G1dr/l using the existing Fab arm exchange efficiency improvement modification reported by Labrijn et al. was 95.6%. At this time, in the case of G1dP3/N3 in which D399K was introduced into one homo body and K409D was introduced into the other homo body, 92.8%, D356K/D399K in one homo body and K409D/K439E in the other homo body were introduced in the case of G1dP5/N5. is 100%, and in addition to the K409R modification of the IgG4 type, the production rate of the bispecific antibody of G1drP1/N1 having D356K in one homo body and K439E in the other homo body in addition to the K409R modification was 99.7%.

0.5 mM GSH의 환원 조건하에서는 기존의 Fab Arm Exchange 효율 향상 개변을 사용한 G1dr/l의 이중특이성 항체 생성률은 9.5%였다. 이때 한쪽 호모체에 D399K를, 다른 한쪽 호모체에 K409D를 도입한 G1dP3/N3의 경우는 4.8%, 한쪽 호모체에 D356K/D399K를 다른 한쪽 호모체에 K409D/K439E를 도입한 G1dP5/N5의 경우는 75.4%, 또한 IgG4형의 K409R 개변에 더하여 한쪽 호모체에 D356K를 다른 한쪽 호모체에 K439E를 갖는 G1drP1/N1의 이중특이성 항체 생성률은 21.8%가 되어, 모두 다른 환원 조건과 비교하여 대폭 저하되었다. Under the reducing conditions of 0.5 mM GSH, the production rate of the G1dr/l bispecific antibody using the existing Fab Arm Exchange efficiency improvement modification was 9.5%. At this time, in the case of G1dP3/N3 in which D399K was introduced into one homo body and K409D was introduced into the other homo body, 4.8%, D356K/D399K in one homo body and K409D/K439E in the other homo body in the case of G1dP5/N5 were introduced. is 75.4%, and the production rate of the G1drP1/N1 bispecific antibody with D356K in one homo body and K439E in the other homo body in addition to the K409R modification of the IgG4 type was 21.8%, and all were significantly lowered compared to other reducing conditions. .

이상의 결과로부터, 어느 반응 조건하에 있어서도 한쪽 호모체에 D356K/D399K를, 다른 한쪽 호모체에 K409D/K439E를 도입한 G1dP5/N5는 Labrijn 등에 의해 보고되어 있는 기존의 Fab Arm Exchange 효율 향상 개변보다도 높은 이중특이성 항체 생성률을 나타내는 것이 명확해졌다. 높은 이중특이성 항체 생성률은 실제로 의약품으로서의 이중특이성 항체의 제조에 있어서 매우 중요하여, 본 개변은 기존의 개변과 비교하여 유용성이 높은 것으로 생각된다. From the above results, under any reaction conditions, G1dP5/N5 in which D356K/D399K was introduced into one homo body and K409D/K439E was introduced into the other homo body under any reaction conditions was higher than the existing Fab Arm Exchange efficiency improvement modification reported by Labrijn et al. It became clear that the specific antibody production rate was shown. A high bispecific antibody production rate is actually very important in the production of a bispecific antibody as a pharmaceutical, and this modification is considered to be highly useful compared to the existing modification.

〔실시예 4〕CH3 도메인 불안정화 개변을 이용한 고효율 Fab Arm Exchange의 개발[Example 4] Development of high-efficiency Fab Arm Exchange using CH3 domain destabilization modification

지금까지의 실시예에 있어서, 회합 계면 제어 개변에 의해 각각의 호모체에 다른 차지의 전하를 도입함으로써 환원제에 의해 생성된 반분자가 우선적으로 다른 호모체 유래의 반분자와 결합하여 높은 효율로 이중특이성 항체가 생성되는 것이 나타내어졌다. 한편 Fab Arm Exchange에 의해 이중특이성 항체가 생성되는 과정에 있어서는, 2종류의 호모체가 환원제로 개열된 후에 CH3 도메인이 해리되어 반분자(HL분자)를 생성하는 과정이 율속단계가 되는 것이 보고되어 있다(Rispens T et al. J. Am. Chem. Soc., 2011. 133. 10302-10311). 즉, 각각의 호모체의 CH3 도메인을 과도하게 불안정화함으로써 CH3 도메인의 해리를 촉진시킬 수 있다면 보다 효율적으로 Fab Arm Exchange를 야기할 수 있을 것으로 기대된다. 이에 먼저 표 2, 표 3에 나타낸 5 mM GSH 존재하에서의 이중특이성 항체 생성률과, 각각의 호모체의 CH3 도메인의 안정성의 관계를 평가하였다. CH3 도메인의 안정성은 참고예 3의 방법에 따라 측정한 Tm(열변성 중간온도)을 지표로 하였다. In the examples so far, by introducing a charge of a different charge to each homo body by modifying the association interface control, the half-molecules generated by the reducing agent preferentially bind with the half-molecules derived from the other homo bodies, resulting in a double-layer with high efficiency. It has been shown that specific antibodies are generated. On the other hand, in the process of generating a bispecific antibody by Fab Arm Exchange, it has been reported that the process of generating a half molecule (HL molecule) by dissociating the CH3 domain after cleavage of two types of homoforms with a reducing agent is the rate-limiting step. (Rispens T et al. J. Am. Chem. Soc., 2011. 133. 10302-10311). That is, if the dissociation of the CH3 domain can be promoted by excessively destabilizing the CH3 domain of each homogenous body, it is expected that Fab arm exchange can be caused more efficiently. Therefore, first, the relationship between the bispecific antibody production rate in the presence of 5 mM GSH shown in Tables 2 and 3 and the stability of the CH3 domain of each homoform was evaluated. The stability of the CH3 domain was measured using the Tm (intermediate temperature of heat denaturation) measured according to the method of Reference Example 3 as an index.

이중특이성 항체 생성률과 사용한 2종류의 호모체 중 CH3의 Tm이 높은 쪽의 값의 관계를 도 3에 나타낸다. 이중특이성 항체 생성률이 낮았던 G1dP1/N1이나 G1dP4/N4의 경우는 CH3의 Tm이 76℃ 이상으로 높았던 한편, 반응 효율이 높은 G1dP5/N5, G1dP3/N3, G1drP1/N1에서의 호모체의 CH3의 Tm은 각각 65.1℃, 69.6℃, 69.5℃였다. 이상의 결과로부터 Fab Arm Exchange에서의 이중특이성 항체 생성률과, 호모체의 CH3의 안정성에는 명확하게 상관관계가 있는 것이 나타내어졌다. 또한 높은 반응 효율을 달성하기 위해서는 사용하는 2종류의 호모체 중 안정한 쪽 호모체의 CH3의 Tm이 70℃ 등을 밑돌도록 CH3영역의 안정성을 불안정하게 하는 것이 바람직한 것이 나타내어졌다. 여기서 같은 조건에서 측정한 천연형 인간 IgG1의 서열을 갖는 MRAH-G1d/MRAL-k0의 CH3영역의 Tm은 83.6℃였던 것으로부터, Fab Arm Exchange에 있어서 높은 반응 효율을 달성하기 위해서는 천연형 인간 IgG1으로부터 CH3영역의 Tm을 예를 들면 13℃ 이상 저하시키도록, CH3영역의 안정성을 불안정하게 할 필요가 있는 것을 알 수 있었다. Fig. 3 shows the relationship between the bispecific antibody production rate and the value of the higher CH3 Tm among the two types of homoforms used. In the case of G1dP1/N1 or G1dP4/N4, which had low bispecific antibody production rates, the Tm of CH3 was as high as 76°C or higher, while the Tm of the homozygous CH3 in G1dP5/N5, G1dP3/N3, and G1drP1/N1 with high reaction efficiency. were 65.1 °C, 69.6 °C, and 69.5 °C, respectively. From the above results, it was shown that there is a clear correlation between the bispecific antibody production rate in Fab Arm Exchange and the stability of homozygous CH3. In addition, it has been shown that in order to achieve high reaction efficiency, it is preferable to destabilize the stability of the CH3 region so that the Tm of CH3 of the stable one of the two types of homoforms used is less than 70°C or the like. Here, the Tm of the CH3 region of MRAH-G1d/MRAL-k0 having the sequence of native human IgG1 measured under the same conditions was 83.6°C. It was found that it was necessary to make the stability of the CH3 region unstable in order to decrease the Tm of the CH3 region by, for example, 13 DEG C or more.

이에 다음으로 사용하는 호모체의 CH3 도메인의 Tm을 저하시킴으로써 이중특이성 항체 생성률이 향상되는지 여부에 대해서 검토하였다. CH3의 안정성을 저하시키는 개변으로서는 IgG2형의 개변인 V397M을 사용하였다. IgG2에서는 EU 넘버링 397번째 아미노산 잔기가 Met가 되어 있어, 이를 IgG1형의 Val로 치환하는 개변을 도입함으로써 CH3영역의 안정성(Tm)이 향상되는 것이 보고되어 있다(WO2009/041613). 따라서 IgG1형의 CH3 도메인에 대해 V397M 개변을 도입함으로써 CH3 도메인이 불안정화되어 해리되기 쉬워질 것으로 기대되었다. Accordingly, it was examined whether the bispecific antibody production rate was improved by lowering the Tm of the CH3 domain of the homoform to be used. As a modification to lower the stability of CH3, V397M, which is an IgG2 type modification, was used. In IgG2, the EU numbering 397th amino acid residue is Met, and it has been reported that the stability (Tm) of the CH3 region is improved by introducing a modification to replace it with Val of IgG1 type (WO2009/041613). Therefore, it was expected that the CH3 domain would be destabilized and easily dissociated by introducing the V397M modification to the CH3 domain of the IgG1 type.

이에 표 2에 있어서 이중특이성 항체 생성률이 낮았던 G1dP1/N1, G1dP4/N4, G1dP6/N6에 대해 양쪽 호모체에 V397M 개변을 도입한 G1dP8/N8, G1dP9/N9, G1dP10/N10을 검토하였다. 구체적으로는, MRAH-G1dP1, MRAH-G1dP4, MRAH-G1dP6, H54-G1dN1, H54-G1dN4, H54-G1dN6에 대해 V397M을 도입한 MRAH-G1dP8(서열번호 29), MRAH-G1dP9(서열번호 30), MRAH-G1dP10(서열번호 31), H54-G1dN8(서열번호 32), H54-G1dN9(서열번호 33), H54-G1dN10(서열번호 34)을 제작하였다. H쇄 가변영역이 MRAH인 경우는 MRAL-k0를, H54인 경우는 L28-k0를 항체 L쇄로서 사용하고, 참고예 1의 방법에 따라 MRAH-G1dP8/MRAL-k0, H54-G1dN8/L28-k0, MRAH-G1dP9/MRAL-k0, H54-G1dN9/L28-k0, MRAH-G1dP10/MRAL-k0, H54-G1dN10/L28-k0를 발현, 정제하였다. 또한 참고예 3의 방법에 따라 얻어진 항체의 Tm을 측정하였다. Therefore, in Table 2, G1dP1/N1, G1dP4/N4, G1dP6/N6, which had low bispecific antibody production rates, G1dP8/N8, G1dP9/N9, and G1dP10/N10 in which V397M modifications were introduced into both homozygotes were reviewed. Specifically, MRAH-G1dP8 (SEQ ID NO: 29), MRAH-G1dP9 (SEQ ID NO: 30) introduced with V397M against MRAH-G1dP1, MRAH-G1dP4, MRAH-G1dP6, H54-G1dN1, H54-G1dN4, H54-G1dN6 , MRAH-G1dP10 (SEQ ID NO: 31), H54-G1dN8 (SEQ ID NO: 32), H54-G1dN9 (SEQ ID NO: 33), H54-G1dN10 (SEQ ID NO: 34) were prepared. When the H chain variable region is MRAH, MRAL-k0 is used, and when it is H54, L28-k0 is used as the antibody L chain, and according to the method of Reference Example 1, MRAH-G1dP8/MRAL-k0, H54-G1dN8/L28- k0, MRAH-G1dP9/MRAL-k0, H54-G1dN9/L28-k0, MRAH-G1dP10/MRAL-k0, H54-G1dN10/L28-k0 were expressed and purified. In addition, the Tm of the antibody obtained according to the method of Reference Example 3 was measured.

다음으로 얻어진 호모체 2종류를 아래에 나타내는 조합으로 혼합하고, 반응산물을 참고예 2의 방법에 따라 평가하였다. Next, two types of the obtained homo bodies were mixed in the combination shown below, and the reaction product was evaluated according to the method of Reference Example 2.

(1) MRAH-G1dP8/MRAL-k0와 H54-G1dN8/L28-k0(1) MRAH-G1dP8/MRAL-k0 and H54-G1dN8/L28-k0

(2) MRAH-G1dP9/MRAL-k0와 H54-G1dN9/L28-k0(2) MRAH-G1dP9/MRAL-k0 and H54-G1dN9/L28-k0

(3) MRAH-G1dP10/MRAL-k0와 H54-G1dN10/L28-k0(3) MRAH-G1dP10/MRAL-k0 and H54-G1dN10/L28-k0

(4) MRAH-G1dr/MRAL-k0와 H54-G1dl/L28-k0(4) MRAH-G1dr/MRAL-k0 and H54-G1dl/L28-k0

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 0.2 ㎎/㎖, 0.05% Tween 20 (Junsei), [GSH(Sigma)] = 5 mM, 37℃, 24시간. Reaction conditions: [each mAb] = 0.2 mg/ml, 0.05% Tween 20 (Junsei), [GSH (Sigma)] = 5 mM, 37 °C, 24 hours in TBS (Takara, pH 7.6).

얻어진 결과를 표 4에 나타낸다.The obtained result is shown in Table 4.

Figure 112016038698342-pct00004
Figure 112016038698342-pct00004

표 중의 「MoAb1의 CH3의 Tm」이란 MRAH의 가변영역을 갖는 호모체의 CH3의 Tm을, 「MoAb2의 CH3의 Tm」이란 H54의 가변영역을 갖는 호모체의 CH3의 Tm을 나타낸다. In the table, "Tm of CH3 of MoAb1" indicates the Tm of CH3 of the homo form having the MRAH variable region, and "Tm of CH3 of MoAb2" indicates the Tm of CH3 of the homo form having the variable region of H54.

G1dP1/N1 양쪽 호모체에 V397M 개변을 도입한 G1dP8/N8의 경우는 MoAb1의 CH3의 Tm이 6.6℃ 저하되어 70.1℃가, MoAb2의 경우는 4.2℃ 저하되어 70.5℃가 되고, 이중특이성 항체가 1.7%에서 73.2%로 향상되었다. G1dP4/N4 양쪽 호모체에 V397M 개변을 도입한 G1dP9/N9의 경우는 MoAb1의 CH3의 Tm이 1.5℃ 저하되어 67℃가, MoAb2의 경우는 5.5℃ 저하되어 71℃가 되고, 이중특이성 항체가 4.4%에서 67.3%로 향상되었다. G1dP6/N6 양쪽 호모체에 V397M 개변을 도입한 G1dP10/N10의 경우는 MoAb2의 CH3의 Tm에 변화는 없었지만, MoAb1의 CH3의 Tm이 2.2℃ 저하되어 63.8℃가 되고, 이중특이성 항체가 29.3%에서 96.9%로 향상되었다. 이상의 결과로부터, V397M 개변을 가하여 호모체의 CH3의 Tm을 저하시킴으로써 Fab Arm Exchange로의 이중특이성 항체 생성 효율을 향상시킬 수 있는 것이 나타내어졌다. 또한 이때 기존의 Fab Arm Exchange 효율 향상 개변을 사용한 G1dr/l의 이중특이성 항체 생성률은 88.1%로, G1dP10/N10은 이보다도 우수한 이중특이성 항체 생성률을 나타내었다. In the case of G1dP8/N8, in which V397M modifications were introduced into both homo bodies of G1dP1/N1, the Tm of CH3 of MoAb1 decreased by 6.6°C to 70.1°C, and in the case of MoAb2, it decreased by 4.2°C to 70.5°C, and the bispecific antibody was 1.7 % to 73.2%. In the case of G1dP9/N9, in which V397M modification was introduced into both G1dP4/N4 homo bodies, the Tm of CH3 of MoAb1 decreased by 1.5°C to 67°C, and in the case of MoAb2, it decreased by 5.5°C to 71°C, and the bispecific antibody was 4.4 % to 67.3%. In the case of G1dP10/N10, in which V397M modification was introduced into both G1dP6/N6 homo bodies, there was no change in the Tm of CH3 of MoAb2, but the Tm of CH3 of MoAb1 was decreased by 2.2°C to 63.8°C, and the bispecific antibody was 29.3% It improved to 96.9%. From the above results, it was shown that by adding V397M modification to lower the Tm of CH3 of the homo body, the efficiency of generating a bispecific antibody by Fab Arm Exchange can be improved. In addition, at this time, the production rate of the bispecific antibody of G1dr/l using the conventional Fab arm exchange efficiency improvement modification was 88.1%, and that of G1dP10/N10 showed a better bispecific antibody production rate than this.

이에 다음으로 효과가 있었던 EU 넘버링 397번째 주변에 대해 보다 큰 CH3 불안정화 효과가 기대되는 개변을 도입하고, 추가로 이중특이성 항체 생성률이 향상되는지 여부를 검토하였다. 도 4는 보고되어 있는 IgG1의 X선 결정구조 해석 데이터(PDB:3DO3)를 사용하여 CH3 도메인의 EU 넘버링 397번째 주변을 나타낸 것이다. This was followed by introducing a modification expected to have a larger CH3 destabilizing effect around the 397th EU numbering effect, and further reviewing whether the bispecific antibody production rate is improved. Figure 4 shows the EU numbering 397th periphery of the CH3 domain using the reported X-ray crystal structure analysis data of IgG1 (PDB:3DO3).

먼저 A쇄의 D399는 B쇄의 K392와 정전 상호작용하고 있는 것으로 생각되기 때문에, V397M 개변에 더하여 K392를 Asp 또는 Glu로 치환함으로써 양쇄의 정전 반발이 일어나, 양쇄의 상호작용을 보다 불안정화할 수 있는 가능성이 있다. 또한 K392를 측쇄 분지형 아미노산으로 치환함으로써 M397과의 입체장애에 의해 양쇄의 회합을 보다 불안정화할 수 있는 것도 기대된다. 또한 EU 넘버링 397번째 아미노산 잔기를 보다 부피가 큰 아미노산으로 치환함으로써 V397M 개변보다도 더 CH3의 회합을 억제할 수 있는 가능성도 기대되었다. 이상과 같은 관점에서 MRAH-G1dP1 및 H54-G1dN1을 베이스로 하여 표 5에 나타낸 7종류의 항체 H쇄 유전자를 새롭게 제작하였다. First, since D399 of the A chain is thought to be in electrostatic interaction with K392 of the B chain, in addition to V397M modification, by replacing K392 with Asp or Glu, electrostatic repulsion of both chains occurs, which can further destabilize the interaction of both chains. There is a possibility. It is also expected that the association of both chains can be further destabilized by steric hindrance with M397 by substituting K392 with a branched branched amino acid for K392. In addition, by replacing the 397th amino acid residue of EU numbering with a bulkier amino acid, the possibility of inhibiting the association of CH3 more than V397M modification was also expected. In view of the above, seven types of antibody H chain genes shown in Table 5 were newly prepared based on MRAH-G1dP1 and H54-G1dN1.

Figure 112016038698342-pct00005
Figure 112016038698342-pct00005

H쇄 가변영역이 MRAH인 경우는 MRAL-k0를, H54인 경우는 L28-k0를 항체 L쇄로서 사용하였다. 참고예 1의 방법에 따라 MRAH-G1dP14/MRAL-k0, H54-G1dN14/L28-k0, MRAH-G1dP15/MRAL-k0, H54-G1dN15/L28-k0, MRAH-G1dP16/MRAL-k0, H54-G1dN16/L28-k0, MRAH-G1dP17/MRAL-k0, H54-G1dN17/L28-k0, MRAH-G1dP18/MRAL-k0, H54-G1dN18/L28-k0, MRAH-G1dP19/MRAL-k0, H54-G1dN19/L28-k0, MRAH-G1dP20/MRAL-k0, H54-G1dN20/L28-k0를 발현, 정제하였다. 또한 참고예 3의 방법에 따라 얻어진 항체의 Tm을 측정하였다. When the H chain variable region was MRAH, MRAL-k0 was used, and in the case of H54, L28-k0 was used as the antibody L chain. According to the method of Reference Example 1, MRAH-G1dP14/MRAL-k0, H54-G1dN14/L28-k0, MRAH-G1dP15/MRAL-k0, H54-G1dN15/L28-k0, MRAH-G1dP16/MRAL-k0, H54-G1dN16 /L28-k0, MRAH-G1dP17/MRAL-k0, H54-G1dN17/L28-k0, MRAH-G1dP18/MRAL-k0, H54-G1dN18/L28-k0, MRAH-G1dP19/MRAL-k0, H54-G1dN19/L28 -k0, MRAH-G1dP20/MRAL-k0, H54-G1dN20/L28-k0 were expressed and purified. In addition, the Tm of the antibody obtained according to the method of Reference Example 3 was measured.

다음으로 얻어진 호모체 2종류를 아래에 나타내는 조합으로 혼합하고, 반응산물을 참고예 2의 방법에 따라 평가하였다. Next, two types of the obtained homo bodies were mixed in the combination shown below, and the reaction product was evaluated according to the method of Reference Example 2.

(1) MRAH-G1dP14/MRAL-k0와 H54-G1dN14/L28-k0(1) MRAH-G1dP14/MRAL-k0 and H54-G1dN14/L28-k0

(2) MRAH-G1dP15/MRAL-k0와 H54-G1dN15/L28-k0(2) MRAH-G1dP15/MRAL-k0 and H54-G1dN15/L28-k0

(3) MRAH-G1dP16/MRAL-k0와 H54-G1dN16/L28-k0(3) MRAH-G1dP16/MRAL-k0 and H54-G1dN16/L28-k0

(4) MRAH-G1dP17/MRAL-k0와 H54-G1dN17/L28-k0(4) MRAH-G1dP17/MRAL-k0 and H54-G1dN17/L28-k0

(5) MRAH-G1dP18/MRAL-k0와 H54-G1dN18/L28-k0(5) MRAH-G1dP18/MRAL-k0 and H54-G1dN18/L28-k0

(6) MRAH-G1dP19/MRAL-k0와 H54-G1dN19/L28-k0(6) MRAH-G1dP19/MRAL-k0 and H54-G1dN19/L28-k0

(7) MRAH-G1dP20/MRAL-k0와 H54-G1dN20/L28-k0(7) MRAH-G1dP20/MRAL-k0 and H54-G1dN20/L28-k0

(8) MRAH-G1dr/MRAL-k0와 H54-G1dl/L28-k0(8) MRAH-G1dr/MRAL-k0 and H54-G1dl/L28-k0

(9) MRAH-G1dP1/MRAL-k0와 H54-G1dN1/L28-k0(9) MRAH-G1dP1/MRAL-k0 and H54-G1dN1/L28-k0

(10) MRAH-G1dP8/MRAL-k0와 H54-G1dN8/L28-k0(10) MRAH-G1dP8/MRAL-k0 and H54-G1dN8/L28-k0

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 0.2 ㎎/㎖, 0.05% Tween 20 (Junsei), [2-MEA(Sigma)] = 25 mM, 37℃, 24시간. Reaction conditions: [each mAb] = 0.2 mg/ml, 0.05% Tween 20 (Junsei), [2-MEA (Sigma)] = 25 mM, 37 °C, 24 hours in TBS (Takara, pH 7.6).

얻어진 결과를 표 6에 나타낸다.The obtained results are shown in Table 6.

Figure 112016038698342-pct00006
Figure 112016038698342-pct00006

G1dP1/N1 양쪽 호모체에 V397M 개변을 도입한 G1dP8/N8의 경우는 이중특이성 항체가 73.2%였던 것에 대해, 이 양쇄에 K392D를 도입한 G1dP14/N14의 경우는 96.5%, K392E를 도입한 G1dP15/N15의 경우는 96.9%, K392T를 도입한 G1dP18/N18의 경우는 98.9%로 크게 이중특이성 항체 생성률이 향상되었다. 또한 G1dP1/N1에 대해 V397M 대신에 V397F를 도입한 G1dP16/N16의 경우는 96.5%, V397Y를 도입한 G1dP17/N17의 경우는 98%로 V397M보다도 이중특이성 항체 생성률이 향상되었다. 이는 V397M을 포함하는 G1dP8/N8의 MoAb1과 MoAb2의 CH3의 Tm이 각각 70.1℃와 70.5℃인 것에 대해, G1dP16/N16의 경우는 69.1℃와 69.7℃, G1dP17/N17의 경우는 69.2℃와 69.8℃인 것으로부터, 목적하는 바대로 V397M 개변보다도 호모체의 CH3 도메인의 상호작용이 약해져 해리되기 쉬워졌기 때문으로 생각된다. 이때 기존의 Fab Arm Exchange 효율 향상 개변을 사용한 G1dr/l의 이중특이성 항체 생성률은 91.7%이고, G1dP14/N14, G1dP15/N15, G1dP16/N16, G1dP17/N17, G1dP18/N18은 이보다도 우수한 이중특이성 항체 생성률을 나타내었다. In the case of G1dP8/N8 introduced with V397M modifications to both G1dP1/N1 homo bodies, the bispecific antibody was 73.2%, whereas in the case of G1dP14/N14 introduced with K392D in both chains, 96.5%, G1dP15/ K392E was introduced In the case of N15, it was 96.9%, and in the case of G1dP18/N18 introduced with K392T, it was 98.9%, which greatly improved the bispecific antibody production rate. In addition, for G1dP1/N1, G1dP16/N16, in which V397F was introduced instead of V397M, was 96.5%, and in the case of G1dP17/N17, in which V397Y was introduced, 98%, the bispecific antibody production rate was improved compared to V397M. This means that the Tm of CH3 of MoAb1 and MoAb2 of G1dP8/N8 containing V397M is 70.1°C and 70.5°C, respectively, whereas in the case of G1dP16/N16, it is 69.1°C and 69.7°C, and in the case of G1dP17/N17, it is 69.2°C and 69.8°C. It is thought that this is because, as desired, the interaction of the CH3 domain of the homo body becomes weaker and dissociation becomes easier than the V397M modification. At this time, the production rate of the bispecific antibody of G1dr/l using the existing Fab Arm Exchange efficiency improvement modification is 91.7%, and the bispecific antibody G1dP14/N14, G1dP15/N15, G1dP16/N16, G1dP17/N17, G1dP18/N18 is superior to this. The production rate was shown.

의약품 제조로의 응용성을 고려한 경우, 기존의 Fab Arm Exchange 효율 향상 개변보다도 이중특이성 항체 생성률이 높고, 헤테로 성분이 적은 G1dP16/N16(D356K/V397F 및 V397F/K439E) 및 G1dP17/N17(D356K/V397Y 및 V397Y/K439E)은 매우 유용하다. When considering the applicability to pharmaceutical manufacturing, G1dP16/N16 (D356K/V397F and V397F/K439E) and G1dP17/N17 (D356K/V397Y) and G1dP16/N16 (D356K/V397F and V397F/K439E) and G1dP17/N17 (D356K/V397Y) with a higher bispecific antibody production rate than the existing Fab Arm Exchange efficiency improvement modifications and V397Y/K439E) are very useful.

또한 표 3 및 표 6에서 검토한 개변체의 CH3의 안정성과 25 mM의 2MEA를 환원제로서 사용했을 때의 이중특이성 항체 생성률의 관계를 도 5에 나타낸다. 도 5에 나타내는 바와 같이, 사용하는 호모체의 CH3의 안정성과 Fab Arm Exchange에 있어서의 효율은 명백하게 상관관계가 있어, 사용하는 2종류의 호모체 중 CH3의 안정성이 높은 쪽의 호모체의 Tm이 70℃ 등을 밑돌도록 CH3영역의 안정성을 불안정화함으로써 높은 이중특이성 항체 생성률이 달성된다.In addition, the relationship between the stability of CH3 of the variants studied in Tables 3 and 6 and the bispecific antibody production rate when 25 mM 2MEA was used as a reducing agent is shown in FIG. 5 . As shown in Fig. 5, there is a clear correlation between the stability of CH3 of the homo body used and the efficiency in Fab arm exchange, and the Tm of the homo body having the higher CH3 stability among the two types of homo body used is higher. A high bispecific antibody production rate is achieved by destabilizing the stability of the CH3 region to be below 70°C or the like.

〔실시예 5〕반응 시간의 검토[Example 5] Examination of reaction time

실시예 4에 있어서 발견된 G1dP17/N17을 사용하여 반응 시간과 반응 효율의 관계를 검토하였다. The relationship between reaction time and reaction efficiency was examined using G1dP17/N17 found in Example 4.

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 1.0 ㎎/㎖, [2-MEA(Sigma)] = 25 mM, 37℃, 전량 50 ㎕. Reaction conditions: [each mAb] = 1.0 mg/ml, [2-MEA (Sigma)] = 25 mM, 37 °C, total amount 50 µl in TBS (Takara, pH 7.6).

90분, 3시간, 24시간에서 4℃로 냉각한 25 mM MES 완충액(pH 5.0)을 450 ㎕ 첨가하고, 추가로 4℃에 보존함으로써 반응을 정지하였다. 그 후 참고예 2의 방법에 따라 반응 효율을 평가하였다(도 6). The reaction was stopped by adding 450 µL of a 25 mM MES buffer (pH 5.0) cooled to 4°C at 90 minutes, 3 hours, and 24 hours, and further storing at 4°C. Thereafter, the reaction efficiency was evaluated according to the method of Reference Example 2 ( FIG. 6 ).

도 6에 나타내어지는 바와 같이, G1dP17/N17의 이중특이성 항체 생성률은 25 mM 2-MEA 존재하, 90분에서 94.6%, 3시간에서 95.2%, 24시간에서 97.4%가 되어, 90분간의 반응 시간에서 약 95%였다. 이 결과로부터, 편쇄에 K409R의 개변을 다른 쪽 편쇄에 F405L의 개변을 도입한 G1dr/l의 이중특이성 항체 생성률과 비교하여(표 6), 충분히 높은 반응 효율을 나타내는 것이 명확해졌다. As shown in FIG. 6 , the production rate of the bispecific antibody of G1dP17/N17 was 94.6% at 90 minutes, 95.2% at 3 hours, and 97.4% at 24 hours in the presence of 25 mM 2-MEA, and the reaction time for 90 minutes was about 95%. From these results, it was clarified that a sufficiently high reaction efficiency was exhibited in comparison with the production rate of a bispecific antibody of G1dr/l in which K409R modification in one side chain and F405L modification in the other side chain were introduced (Table 6).

〔실시예 6〕고효율 Fab Arm Exchange를 나타내는 개변체의 인간 FcgR 및 인간 FcRn으로의 결합 평가[Example 6] Evaluation of binding to human FcgR and human FcRn of variants exhibiting high-efficiency Fab Arm Exchange

실시예 4에 있어서 고효율 FAE 효율을 나타낸 개변체 G1dP17/N17에 대해서 인간 FcgR 및 인간 FcRn으로의 결합 평가를 실시하였다. 먼저 천연형 IgG1의 서열을 갖는 MRAH-G1d/MRAL-k0 및 Fab-Arm Exchange 후의 개변체 MRAH-G1dP17/MRAL-k0//H54-G1dN17/L28-k0의 인간 FcRn과의 결합을 참고예 5의 방법에 따라 실시하였다. 인간 FcRn으로의 결합 해석 결과를 표 7에 나타낸다.Binding to human FcgR and human FcRn was evaluated for the variant G1dP17/N17 showing high-efficiency FAE efficiency in Example 4. First, MRAH-G1d / MRAL-k0 having the sequence of native IgG1 and the mutant MRAH-G1dP17 / MRAL-k0 / / H54-G1dN17 / L28-k0 after Fab-Arm Exchange binding to human FcRn of Reference Example 5 It was carried out according to the method. Table 7 shows the results of analysis of binding to human FcRn.

Figure 112016038698342-pct00007
Figure 112016038698342-pct00007

표 7에 나타낸 결과로부터 Fab-Arm Exchange로 제작한 개변체 MRAH-G1dP17/MRAL-k0//H54-G1dN17/L28-k0는 인간 FcRn에 대해 천연형 IgG1과 동등한 결합 활성을 갖는 것이 명확해졌다. From the results shown in Table 7, it was clarified that the variant MRAH-G1dP17/MRAL-k0//H54-G1dN17/L28-k0 produced by Fab-Arm Exchange has a binding activity equivalent to that of native IgG1 to human FcRn.

다음으로 인간 FcgR에 대한 결합 활성을 참고예 4의 방법에 따라 평가하였다. 여기에서는 천연형 IgG1의 서열을 갖는 MRAH-G1d/MRAL-k0, Fab-Arm Exchange 후의 개변체 MRAH-G1dP17/MRAL-k0//H54-G1dN17/L28-k0에 더하여 Fab-Arm Exchange 반응 전의 2종류의 호모체(MRAH-G1dP17/MRAL-k0와 H54-G1dN17/L28-k0) 및 CH3 도메인을 불안정화하는 개변인 V397Y를 뺀 2종류의 호모체(MRAH-G1dP1/MRAL-k0와 H54-G1dN1/L28-k0)에 대해서도 함께 평가하였다. 표 8에 있어서 KD fold hFcgRIa, KD fold hFcgRIIaR, KD fold hFcgRIIaH, KD fold hFcgRIIb, KD fold hFcgRIIIaV란, G1d의 각각의 FcgR에 대한 KD를 1로 한 경우의 각 개변체의 상대적 결합 활성을 나타낸 값이다. Next, binding activity to human FcgR was evaluated according to the method of Reference Example 4. Here, in addition to MRAH-G1d/MRAL-k0 having a sequence of native IgG1, mutant MRAH-G1dP17/MRAL-k0//H54-G1dN17/L28-k0 after Fab-Arm Exchange, two types before Fab-Arm Exchange reaction Homoforms of (MRAH-G1dP17/MRAL-k0 and H54-G1dN17/L28-k0) and two types of homoforms (MRAH-G1dP1/MRAL-k0 and H54-G1dN1/L28) except for V397Y, a modification that destabilizes the CH3 domain -k0) was also evaluated. In Table 8, KD fold hFcgRIa, KD fold hFcgRIIaR, KD fold hFcgRIIaH, KD fold hFcgRIIb, KD fold hFcgRIIIaV is a value showing the relative binding activity of each variant when the KD for each FcgR of G1d is 1. .

Figure 112016038698342-pct00008
Figure 112016038698342-pct00008

천연형과 비교하여 Fab-Arm Exchange 후의 개변체 G1dP17/N17은 hFcgRIa로의 결합이 1.1배, hFcgRIIaR로의 결합이 2.7배, hFcgRIIaH로의 결합이 2.3배, hFcgRIIb로의 결합이 2.5배, hFcgRIIIaV로의 결합이 2.5배 증강되어 있었다. 여기에서 CH3 도메인을 불안정화하는 V397Y를 도입하기 전의 호모체 G1dP1 및 G1dN1에서는 어느 FcgR에 대해서도 천연형과 동등한 것, 또한 여기에 V397Y가 도입된 호모체(G1dP17 및 G1dN17)의 경우는 각 FcgR에 대한 결합이 증강되어 있는 것으로부터, V397Y 개변에 의해 hFcgR로의 결합이 증강된 것을 알 수 있다. Compared with the native type, the modified G1dP17/N17 after Fab-Arm Exchange had 1.1-fold binding to hFcgRIa, 2.7-fold binding to hFcgRIIaR, 2.3-fold binding to hFcgRIIaH, 2.5-fold binding to hFcgRIIb, 2.5-fold binding to hFcgRIIIaV. was augmented. Here, in the homozygous G1dP1 and G1dN1 before the introduction of V397Y destabilizing the CH3 domain, it is equivalent to the native type for any FcgR, and in the case of the homogenous body into which V397Y is introduced (G1dP17 and G1dN17), binding to each FcgR From this enhancement, it can be seen that the binding to hFcgR was enhanced by the V397Y modification.

이상의 결과로부터, 높은 Fab-Arm Exchange 효율을 발현하는 G1dP17/N17은 천연형과 비교하여 인간 FcRn, 인간 FcgR에 대한 결합을 손상시키지 않는 것이 나타내어졌다. From the above results, it was shown that G1dP17/N17 expressing high Fab-Arm Exchange efficiency does not impair binding to human FcRn and human FcgR compared with the native type.

〔실시예 7〕배양상청 중에 있어서의 Fab-Arm Exchange의 검토[Example 7] Review of Fab-Arm Exchange in the culture supernatant

Fab-Arm Exchange에 의한 이중특이성 항체의 제조에서는, 2종류의 호모체를 따로 따로 배양, 정제한 후에 Fab-Arm Exchange를 행하는 것이 상정되는데, 만일 배양상청 중에서 반응을 일으킬 수 있다면 호모체의 정제단계를 생략할 수 있기 때문에 커다란 이점이 된다. 이에 2종류의 호모체를 배양상청 중에서 환원제와 함께 혼합하여, Fab-Arm Exchange가 높은 효율로 일어나는지 여부를 검증하였다. In the production of bispecific antibodies by Fab-Arm Exchange, it is assumed that Fab-Arm Exchange is performed after separately culturing and purifying two types of homo bodies. can be omitted, which is a huge advantage. Accordingly, by mixing two types of homo bodies with a reducing agent in the culture supernatant, it was verified whether Fab-Arm Exchange occurs with high efficiency.

먼저 MRAH-G1d 및 H54-G1d의 356번째 아미노산 잔기를 E로 358번째 아미노산 잔기를 M으로 치환한 MRAH-G1m(서열번호 49), H54-G1m(서열번호 50)을 각각 제작하였다. 다음으로 MRAH-G1m에 대해 E356K 및 K409R을 도입한 MRAH-G1mrP1(서열번호 51), H54-G1m에 대해 K439E 및 K409R을 도입한 H54-G1mrN1(서열번호 52)을 제작하였다. H쇄 가변영역이 MRAH인 경우는 MRAL-k0를, H54인 경우는 L28-k0를 항체 L쇄로서 사용하였다. 참고예 1의 방법에 따라 MRAH-G1mrP1/MRAL-k0, H54-G1mrN1/L28-k0를 발현, 정제하였다.First, MRAH-G1m (SEQ ID NO: 49) and H54-G1m (SEQ ID NO: 50) in which the 356th amino acid residue of MRAH-G1d and H54-G1d were substituted with E and the 358th amino acid residue with M were prepared, respectively. Next, MRAH-G1mrP1 (SEQ ID NO: 51) introduced with E356K and K409R for MRAH-G1m, and H54-G1mrN1 (SEQ ID NO: 52) introduced with K439E and K409R for H54-G1m were prepared. When the H chain variable region was MRAH, MRAL-k0 was used, and in the case of H54, L28-k0 was used as the antibody L chain. MRAH-G1mrP1/MRAL-k0 and H54-G1mrN1/L28-k0 were expressed and purified according to the method of Reference Example 1.

또한 FreeStyle293세포(Invitrogen)를 FreeStyle 293 Expression medium에서 배양한 후, 원심하여 상청을 회수하고 0.22 ㎛의 여과막으로 여과한 것을 MocK CM으로서 Fab-Arm Exchange에 사용하였다. In addition, FreeStyle 293 cells (Invitrogen) were cultured in FreeStyle 293 Expression medium, centrifuged to recover the supernatant, and filtered through a 0.22 µm filtration membrane was used as MocK CM for Fab-Arm Exchange.

반응 조건：Mock CM(pH 7.6) 중 [각 mAb] = 1.0 ㎎/㎖, [2-MEA(Sigma)] = 25 mM, 37℃, 90분Reaction conditions: [each mAb] = 1.0 mg/ml, [2-MEA (Sigma)] = 25 mM, 37 °C, 90 min in Mock CM (pH 7.6)

반응 후의 반응용액에 rProtein A Sepharose Fast Flow(GE 헬스케어)를 첨가하여 정제한 후에, 참고예 2의 방법에 따라 반응 효율을 평가하였다(도 7). After the reaction solution was purified by adding rProtein A Sepharose Fast Flow (GE Healthcare) to the reaction solution, the reaction efficiency was evaluated according to the method of Reference Example 2 (FIG. 7).

도 7에 나타낸 바와 같이, 배양상청 중이라도 25 mM 2-MEA 존재하, 37℃, 90분간 반응시킴으로써 98% 이상의 반응 효율로 이중특이성 항체가 생성되는 것이 명확해졌다. As shown in FIG. 7 , it was clarified that the bispecific antibody was produced with a reaction efficiency of 98% or more by reacting in the presence of 25 mM 2-MEA in the culture supernatant at 37° C. for 90 minutes.

〔실시예 8〕마우스 IgG1에서의 Fab-Arm Exchange의 개발[Example 8] Development of Fab-Arm Exchange in mouse IgG1

지금까지의 실시예에 있어서, 인간 IgG1 또는 인간 IgG4에서 효율적으로 Fab-Arm Exchange가 야기되는 것이 나타내어졌다. 다음으로 마우스 IgG1에서도 동일하게 Fab-Arm Exchange에 의해 이중특이성 항체가 생성되는지 여부를 검증하였다. In the examples so far, it has been shown that Fab-Arm Exchange is efficiently induced in human IgG1 or human IgG4. Next, it was verified whether the bispecific antibody was generated by Fab-Arm Exchange in the same way in mouse IgG1.

보고되어 있는 결정구조(Harris LJ et al. J. Mol. Biol., 1998. 275. 861-872)로부터, EU 넘버링 399번째 D와 409번째 K가 CH3 도메인의 사슬 간 상호작용에 기여하고 있는 것으로 추측되었다(도 8). 이에 그 부위에 대해 인간 IgG1과 마찬가지로 헤테로 이량화를 촉진하기 위한 전하를 도입하여 Fab-Arm Exchange가 야기되는지 여부를 검증하였다. From the reported crystal structure (Harris LJ et al. J. Mol. Biol., 1998. 275. 861-872), EU numbering 399 D and 409 K contribute to the interchain interaction of the CH3 domain. guessed (Fig. 8). Accordingly, it was verified whether Fab-Arm Exchange was caused by introducing a charge to promote heterodimerization to the site as in human IgG1.

항체 H쇄 가변영역에는 WO2009/125825에 개시되어 있는 인간 인터류킨 6 수용체에 대한 항체의 H쇄 가변영역인 WT(H)(서열번호 1：이후 MRAH로 기재) 및 H54(서열번호 2)를 사용하였다. 이들 H쇄 가변영역에 대해 항체 H쇄 불변영역으로서 마우스 IgG1의 H쇄 불변영역을 갖는 MRAH-mIgG1(서열번호 53) 및 H54-mIgG1(서열번호 54)을 제작하였다. 또한 회합 계면 제어 개변으로서 MRAH-mIgG1에 D399K를 도입한 MRAH-mIgG1mP3(서열번호 55), D399R을 도입한 MRAH-mIgG1mP4(서열번호 56), H54-mIgG1에 K409D를 도입한 H54-mIgG1mN3(서열번호 57), K409E를 도입한 H54-mIgG1mN4(서열번호 58)를 제작하였다. L쇄로서 마우스 κ쇄 불변영역의 서열을 갖는 MRAL-mk1(서열번호 59) 및 L28-mk1(서열번호 60)을 제작하고, H쇄 가변영역이 MRAH인 경우는 MRAL-mk1을, H54인 경우는 L28-mk1을 항체 L쇄로서 사용하였다. 참고예 1의 방법에 따라 MRAH-mIgG1mP3/MRAL-mk1, MRAH-mIgG1mP4/MRAL-mk1, H54-mIgG1mN3/L28-mk1, H54-mIgG1mN4/L28-mk1을 발현, 정제하였다.For the antibody H chain variable region, WT (H) (SEQ ID NO: 1: hereinafter referred to as MRAH) and H54 (SEQ ID NO: 2), which are the H chain variable regions of the antibody against human interleukin 6 receptor disclosed in WO2009/125825 were used. . For these H chain variable regions, MRAH-mIgG1 (SEQ ID NO: 53) and H54-mIgG1 (SEQ ID NO: 54) having an H chain constant region of mouse IgG1 were prepared as antibody H chain constant regions. In addition, as association interface control modifications, MRAH-mIgG1mP3 (SEQ ID NO: 55) introduced with D399K into MRAH-mIgG1, MRAH-mIgG1mP4 with D399R (SEQ ID NO: 56), H54-mIgG1mN3 (SEQ ID NO: 56) with K409D introduced into H54-mIgG1 57), to which K409E was introduced, H54-mIgG1mN4 (SEQ ID NO: 58) was prepared. MRAL-mk1 (SEQ ID NO: 59) and L28-mk1 (SEQ ID NO: 60) having the sequence of the mouse κ chain constant region as the L chain were prepared, and when the H chain variable region is MRAH, MRAL-mk1, and when H54 used L28-mk1 as the antibody L chain. According to the method of Reference Example 1, MRAH-mIgG1mP3/MRAL-mk1, MRAH-mIgG1mP4/MRAL-mk1, H54-mIgG1mN3/L28-mk1, H54-mIgG1mN4/L28-mk1 were expressed and purified.

다음으로 아래의 조합으로 Fab-Arm Exchange를 실시하였다. Next, Fab-Arm Exchange was performed in the following combination.

(1) MRAH-mIgG1mP3/MRAL-mk1과 H54-mIgG1mN3/L28-mk1(1) MRAH-mIgG1mP3/MRAL-mk1 and H54-mIgG1mN3/L28-mk1

(2) MRAH-mIgG1mP4/MRAL-mk1과 H54-mIgG1mN4/L28-mk1(2) MRAH-mIgG1mP4/MRAL-mk1 and H54-mIgG1mN4/L28-mk1

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 2 ㎎/㎖, [2-MEA(Sigma)] = 25 mM, 37℃, 19시간. Reaction conditions: [each mAb] = 2 mg/ml, [2-MEA (Sigma)] = 25 mM, 37 °C, 19 hours in TBS (Takara, pH 7.6).

반응 후, 참고예 5의 방법에 따라 CE-IEF에 의해 반응 효율을 결정하였다(도 9). After the reaction, the reaction efficiency was determined by CE-IEF according to the method of Reference Example 5 (FIG. 9).

그 결과, MRAH-mIgG1mP3/MRAL-mk1과 H54-mIgG1mN3/L28-mk1을 반응시킨 경우에서는 89.2%, MRAH-mIgG1mP4/MRAL-mk1과 H54-mIgG1mN4/L28-mk1의 경우는 89.9%로 모두 높은 효율로 이중특이성 항체가 생성되고 있는 것이 확인되었다. 인간 IgG1 또는 인간 IgG4를 사용한 Fab-Arm Exchange와 비교하여 반응 효율이 약간 저하되는 것은 마우스 IgG1의 경우는 힌지영역의 디설피드 결합이 3개 존재하여 인간 IgG1 또는 인간 IgG4의 힌지보다도 2개의 중쇄가 보다 강고하게 결합되어 있기 때문으로 예상된다(Harris LJ et al. J. Mol. Biol., 1998. 275. 861-872).As a result, in the case of reacting MRAH-mIgG1mP3/MRAL-mk1 and H54-mIgG1mN3/L28-mk1, 89.2%, and in the case of MRAH-mIgG1mP4/MRAL-mk1 and H54-mIgG1mN4/L28-mk1, the efficiency was 89.9%. It was confirmed that the bispecific antibody was being generated. Compared to the Fab-Arm Exchange using human IgG1 or human IgG4, the reaction efficiency is slightly lowered. In the case of mouse IgG1, there are three disulfide bonds in the hinge region, so two heavy chains are more than those of human IgG1 or human IgG4. It is expected because it is strongly bound (Harris LJ et al. J. Mol. Biol., 1998. 275. 861-872).

〔실시예 9〕마우스 IgG1의 Fab Arm Exchange로 제작한 이중특이성 항체의 마우스 FcgR 및 마우스 FcRn에 대한 결합 활성의 평가[Example 9] Evaluation of binding activity to mouse FcgR and mouse FcRn of a bispecific antibody prepared by Fab Arm Exchange of mouse IgG1

참고예 4-3에 따라 마우스 IgG형 Fab-Arm Exchange로 제작한 2종류의 이중특이성 항체(MRAH-mIgG1mP3/MRAL-mk1//H54-mIgG1mN3/L28-mk1 및 MRAH-mIgG1mP4/MRAL-mk1//H54-mIgG1mN4/L28-mk1)의 마우스 FcgR 및 마우스 FcRn으로의 결합을 평가하였다. 또한 MRAH-mIgG1/MRAL-mk1을 참고예 1에 따라 제작하여 비교 대상으로서 측정하였다. 측정 결과를 표 9에 나타낸다.Two types of bispecific antibodies (MRAH-mIgG1mP3/MRAL-mk1//H54-mIgG1mN3/L28-mk1 and MRAH-mIgG1mP4/MRAL-mk1// Binding of H54-mIgG1mN4/L28-mk1) to mouse FcgR and mouse FcRn was evaluated. In addition, MRAH-mIgG1/MRAL-mk1 was prepared according to Reference Example 1 and measured as a comparison object. Table 9 shows the measurement results.

Figure 112016038698342-pct00009
Figure 112016038698342-pct00009

제작한 2종류의 이중특이성 항체는 모두 천연형 mIgG1과 동일한 결합 프로파일을 나타내었다. 구체적으로는, mFcgRI 및 mFcgRIV에는 결합하지 않고, mFcgRII 및 mFcgRIII에 대해서는 천연형 mIgG1과 비교하여 1.2배의 결합 활성을 나타내었다. Both of the prepared bispecific antibodies exhibited the same binding profile as the native mIgG1. Specifically, it did not bind to mFcgRI and mFcgRIV, and exhibited 1.2-fold binding activity to mFcgRII and mFcgRIII compared with native mIgG1.

다음으로 참고예 4-4에 따라 mFcRn으로의 결합을 평가한 결과를 표 10에 나타낸다.Next, Table 10 shows the results of evaluation of binding to mFcRn according to Reference Example 4-4.

Figure 112016038698342-pct00010
Figure 112016038698342-pct00010

제작한 2종류의 이중특이성 항체는 모두 mFcRn에 대해 천연형 mIgG1과 동등한 결합을 유지하고 있는 것이 나타내어졌다. It was shown that both of the prepared bispecific antibodies maintained the binding equivalent to native mIgG1 to mFcRn.

〔실시예 10〕세포상해활성의 측정[Example 10] Measurement of cytotoxic activity

Fab-Arm Exchange에 의해 제작된 인간 IgG형 이중특이성 항체, 마우스 IgG형 이중특이성 항체가 기존의 수법에 의해 제작된 이중특이성 항체와 동등한 기능을 유지하고 있는지 여부를, 항인간 글리피칸 3 및 항인간 CD3의 이중특이성 항체에 의한 세포상해활성을 측정함으로써 평가하였다. 먼저 대조로서 Schaefer 등에 의해 보고되어 있는 CrossMab 기술(Proc Natl Acad Sci, 2011, 108, 11187-11192)에 의해 인간 IgG4의 불변영역을 갖는 항인간 GPC3/항인간 CD3 이중특이성 항체가 제작되었다. CrossMab 기술에 의해 제작된 본 분자는 WO2012/073985에 기재되어 있는 인간 GPC3에 대한 Fab의 VH 도메인과 VL 도메인이 치환된 분자가 되어 있다. 또한 항체 H쇄 불변영역에는 헤테로 회합을 촉진하기 위해 Knobs-into-Holes 기술을 사용하였다. Knobs-into-Holes 기술은 한쪽 H쇄의 CH3영역에 존재하는 아미노산 측쇄를 보다 큰 측쇄(Knob;돌기)로 치환하고, 다른 한쪽 H쇄의 CH3영역에 존재하는 아미노산 측쇄를 보다 작은 측쇄(Hole;공극)로 치환함으로써 돌기가 공극 내에 배치되도록 하여 H쇄의 헤테로 이량화를 촉진하여 목적의 헤테로 이량화 항체를 효율적으로 취득할 수 있는 기술이다(Nature, 1994, 372, 379-383). 또한 FcgR에 대한 결합을 감약시키는 개변으로서 WO2011/108714에 기재되어 있는 개변이 사용되었다. 구체적으로는 EU 넘버링 234번째, 235번째, 297번째 아미노산 잔기를 Ala로 치환하는 개변이 도입되었다. 항체 H쇄의 C말단으로부터는 EU 넘버링 446번째 Gly 및 447번째 Lys를 제거하고, 이에 대해 추가로 항체 발현 후의 정제를 용이하게 하기 위해 항인간 GPC3측 H쇄의 C말단에 히스티딘 태그를, 항인간 CD3측 H쇄의 C말단에 FLAG 태그를 부가하였다. 이상의 개변을 도입한 항인간 GPC3측 H쇄로서 GC33(2)H-G4dKnHS(서열번호 61)가 제작되었다. 또한 항인간 CD3측 H쇄로서 rCE115H-G4dHlFS(서열번호 62)가 제작되었다. 항체 L쇄로서는 항인간 GPC3측으로서 GC33(2)L-k0(서열번호 63)를, 항인간 CD3측으로서 rCE115L-k0(서열번호 64)를 사용하였다. 이들 항체의 발현은 참고예 1에 따라 FreeStyle293 세포에서 일과성 발현시켰다. 얻어진 배양상청이 MabSelect SuRe 칼럼(GE Healthcare사)에 첨가되고, 당해 칼럼의 세정 후, 50 mM 초산에 의한 용출이 실시되었다. 항체를 포함하는 분획이 HisTrap HP 칼럼(GE Healthcare사) 또는 Ni Sepharose FF 칼럼(GE Healthcare사)에 첨가되고, 당해 칼럼의 세정 후, 이미다졸에 의한 용출이 실시되었다. 항체를 포함하는 분획이 한외여과막으로 농축된 후, 농축액이 Superdex 200 칼럼(GE Healthcare사)에 첨가되고, 그 용출액 단량체의 항체만을 회수함으로써 정제 항체 GPC3 ERY22-rCE115가 얻어졌다.Whether the human IgG-type bispecific antibody and the mouse IgG-type bispecific antibody produced by Fab-Arm Exchange maintain the same functions as the bispecific antibody produced by conventional methods, anti-human glypican 3 and anti-human It was evaluated by measuring the cytotoxic activity by the bispecific antibody of CD3. First, as a control, an anti-human GPC3/anti-human CD3 bispecific antibody having a human IgG4 constant region was prepared by CrossMab technology (Proc Natl Acad Sci, 2011, 108, 11187-11192) reported by Schaefer et al. This molecule produced by CrossMab technology is a molecule in which the VH domain and the VL domain of the Fab for human GPC3 described in WO2012/073985 are substituted. In addition, the Knobs-into-Holes technology was used to promote hetero-association in the antibody H chain constant region. The Knobs-into-Holes technology replaces the amino acid side chain in the CH3 region of one H chain with a larger side chain (Knob; protrusion), and replaces the amino acid side chain in the CH3 region of the other H chain with a smaller side chain (Hole; It is a technology that can efficiently obtain the target heterodimerization antibody by promoting heterodimerization of the H chain by displacing the protrusions within the pores by substituting with a void) (Nature, 1994, 372, 379-383). Also, modifications described in WO2011/108714 were used as modifications to reduce binding to FcgR. Specifically, EU numbering 234th, 235th, 297th amino acid residues were introduced with Ala substitution. From the C-terminus of the antibody H chain, EU numbering 446th Gly and 447th Lys are removed, and a histidine tag is added to the C terminus of the anti-human GPC3 side H chain to facilitate purification after antibody expression, anti-human A FLAG tag was added to the C terminus of the H chain on the CD3 side. GC33(2)H-G4dKnHS (SEQ ID NO: 61) was prepared as an anti-human GPC3 side H chain into which the above modifications were introduced. In addition, rCE115H-G4dHlFS (SEQ ID NO: 62) was prepared as an anti-human CD3 side H chain. As the antibody L chain, GC33(2)L-k0 (SEQ ID NO: 63) was used as the anti-human GPC3 side and rCE115L-k0 (SEQ ID NO: 64) was used as the anti-human CD3 side. Expression of these antibodies was transiently expressed in FreeStyle293 cells according to Reference Example 1. The obtained culture supernatant was added to a MabSelect SuRe column (GE Healthcare), and the column was washed, followed by elution with 50 mM acetic acid. The antibody-containing fraction was added to a HisTrap HP column (GE Healthcare) or a Ni Sepharose FF column (GE Healthcare), and after washing the column, elution with imidazole was performed. After the antibody-containing fraction was concentrated with an ultrafiltration membrane, the concentrate was added to a Superdex 200 column (GE Healthcare), and only the antibody of the eluate monomer was recovered to obtain a purified antibody GPC3 ERY22-rCE115.

다음으로 Fab Arm Exchange에 의해 인간 IgG1형, 인간 IgG4형, 마우스 IgG1형의 불변영역을 갖고, 가변영역이 항인간 GPC3/항인간 CD3인 이중특이성 항체가 제작되었다. 인간 IgG1형 및 인간 IgG4형의 H쇄 불변영역에는 Fab Arm Exchange용 개변을 포함하는 G1dP17, G1dN17, G1drP1, G1drN1, G4dP1, G4dN1에 대해 FcgR 결합 저감 개변으로서 EU 넘버링 235번째 아미노산 잔기를 Arg로 239번째 아미노산 잔기를 Lys로 치환하는 개변을 도입한 F760P17, F760N17, F760G1drP1, F760G1drN1, F760G4dP1, F760G4dN1이 각각 제작되었다. 마우스 IgG1형의 H쇄 불변영역에는 실시예 8에서 사용한 mIgG1mP4, mIgG1mN4에 대해 FcgR 결합 저감 개변으로서 EU 넘버링 235번째, 239번째 아미노산 잔기를 Lys로 치환하는 개변을 도입한 mF18mP4, mF18mN4가 제작되었다. 가변영역으로서는 WO2012/073985에 기재되어 있는 항인간 GPC3의 서열을 사용하여, H0000-F760N17(서열번호 65), H0000-F760G1drN1(서열번호 66), H0000-F760G4dN1(서열번호 67), H0000-mF18mN4(서열번호 68)가 제작되었다. 한편 인간 CD3측 H쇄로서 rCE115H-F760P17(서열번호 69), rCE115H-F760G1drP1(서열번호 70), rCE115H-F760G4dP1(서열번호 71), rCE115H-mF18mP4(서열번호 72)가 제작되었다. 인간 IgG1형 및 인간 IgG4형의 항체 L쇄로서는 항인간 GPC3측으로서 GL4-k0(서열번호 79)를, 항인간 CD3측으로서 rCE115L-k0(서열번호 64)를 공통으로 사용하였다. 또한 마우스 IgG1형의 항체 L쇄로서는 항인간 GPC3측으로서 GL4-mk1(서열번호 80)을, 항인간 CD3측으로서 rCE115L-mk1(서열번호 81)을 사용하였다. 이들 호모체를 참고예 1의 방법에 따라 발현, 정제하여 rCE115H-F760P17/rCE115L-k0, H0000-F760N17/GL4-k0, rCE115H-F760G1drP1/rCE115L-k0, H0000-F760G1drN1/GL4-k0, rCE115H-F760G4dP1/rCE115L-k0, H0000-F760G4dN1/GL4-k0, rCE115H-mF18mP4/rCE115L-mk1, H0000-mF18mP4/GL4-mk1을 얻었다. Next, by Fab Arm Exchange, a bispecific antibody having constant regions of human IgG1 type, human IgG4 type, and mouse IgG1 type and whose variable region is anti-human GPC3/anti-human CD3 was produced. In the H chain constant region of human IgG1 type and human IgG4 type, G1dP17, G1dN17, G1drP1, G1drN1, G4dP1, G4dN1 containing modifications for Fab Arm Exchange as FcgR binding reduction modifications, EU numbering 235th amino acid residue as Arg 239th F760P17, F760N17, F760G1drP1, F760G1drN1, F760G4dP1, F760G4dN1 introduced modifications to replace amino acid residues with Lys were prepared, respectively. In the H chain constant region of mouse IgG1 type, mF18mP4, mF18mN4 introduced in the EU numbering 235th and 239th amino acid residues as changes to reduce FcgR binding to mIgG1mP4, mIgG1mN4 used in Example 8 were introduced with Lys. As the variable region, using the sequence of anti-human GPC3 described in WO2012/073985, H0000-F760N17 (SEQ ID NO: 65), H0000-F760G1drN1 (SEQ ID NO: 66), H0000-F760G4dN1 (SEQ ID NO: 67), H0000-mF18mN4 ( SEQ ID NO: 68) was constructed. Meanwhile, rCE115H-F760P17 (SEQ ID NO: 69), rCE115H-F760G1drP1 (SEQ ID NO: 70), rCE115H-F760G4dP1 (SEQ ID NO: 71), and rCE115H-mF18mP4 (SEQ ID NO: 72) were produced as human CD3 side H chains. As the human IgG1-type and human IgG4-type antibody L chains, GL4-k0 (SEQ ID NO: 79) as the anti-human GPC3 side and rCE115L-k0 (SEQ ID NO: 64) as the anti-human CD3 side were commonly used. As the mouse IgG1-type antibody L chain, GL4-mk1 (SEQ ID NO: 80) was used as the anti-human GPC3 side and rCE115L-mk1 (SEQ ID NO: 81) was used as the anti-human CD3 side. These homoforms were expressed and purified according to the method of Reference Example 1 to rCE115H-F760P17/rCE115L-k0, H0000-F760N17/GL4-k0, rCE115H-F760G1drP1/rCE115L-k0, H0000-F760G1drN1/GL4-k0, rCE115H-F760G4dP1 /rCE115L-k0, H0000-F760G4dN1/GL4-k0, rCE115H-mF18mP4/rCE115L-mk1, H0000-mF18mP4/GL4-mk1 were obtained.

다음으로 얻어진 호모체 2종류를 아래에 나타내는 조합으로 혼합하고, FAE 반응을 일으켰다. Next, the two types of obtained homo bodies were mixed in the combination shown below, and the FAE reaction was raised.

(1) rCE115H-F760P17/rCE115L-k0와 H0000-F760N17/GL4-k0(1) rCE115H-F760P17/rCE115L-k0 and H0000-F760N17/GL4-k0

(2) rCE115H-F760G1drP1/rCE115L-k0와 H0000-F760G1drN1/GL4-k0(2) rCE115H-F760G1drP1/rCE115L-k0 and H0000-F760G1drN1/GL4-k0

(3) rCE115H-F760G4dP1/rCE115L-k0와 H0000-F760G4dN1/GL4-k0(3) rCE115H-F760G4dP1/rCE115L-k0 and H0000-F760G4dN1/GL4-k0

(4) rCE115H-mF18mP4/rCE115L-mk1과 H0000-mF18mP4/GL4-mk1(4) rCE115H-mF18mP4/rCE115L-mk1 and H0000-mF18mP4/GL4-mk1

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 0.36 ㎎/㎖, [2-MEA(Sigma)] = 25 mM, 37℃, 18시간. Reaction conditions: [each mAb] = 0.36 mg/ml, [2-MEA (Sigma)] = 25 mM, 37°C, 18 hours in TBS (Takara, pH 7.6).

반응 후의 산물을 PBS에 투석하고, 세포상해활성의 평가에 사용하였다. The product after the reaction was dialyzed against PBS and used for evaluation of cytotoxic activity.

세포상해활성의 평가는 참고예 6에 기재된 방법으로 실시되었다. 결과를 도 10-1, 도 10-2에 나타낸다.The evaluation of cytotoxic activity was carried out by the method described in Reference Example 6. The results are shown in Figs. 10-1 and 10-2.

도 10-1에 나타낸 바와 같이, 인간 IgG1형 및 인간 IgG4형의 Fab Arm Exchange로 제작된 이중특이성 항체는 모두 기존의 이중특이성 항체 제작기술로 제작된 대조 항체(GPC3 ERY22-rCE115)와 동등한 세포상해활성을 나타내었다. 또한 도 10-2에 나타낸 바와 같이, 마우스 IgG형 Fab Arm Exchange로 제작된 이중특이성 항체도 기존의 이중특이성 항체 제작기술로 제작된 대조 항체(GPC3 ERY22-rCE115)와 동등한 세포상해활성을 나타내었다. As shown in FIG. 10-1, the bispecific antibodies produced by Fab Arm Exchange of human IgG1 type and human IgG4 type both cause cytotoxicity equivalent to that of the control antibody (GPC3 ERY22-rCE115) manufactured by the conventional bispecific antibody production technology. showed activity. In addition, as shown in Fig. 10-2, the bispecific antibody produced by the mouse IgG-type Fab Arm Exchange also exhibited cytotoxic activity equivalent to the control antibody (GPC3 ERY22-rCE115) produced by the conventional bispecific antibody production technology.

〔실시예 11〕정상 마우스 PK 시험[Example 11] Normal mouse PK test

(11-1) 정상 마우스를 사용한 인 비보(in vivo) 시험(11-1) In vivo test using normal mice

인간 IgG형 및 마우스 IgG형 Fab Arm Exchange에 의해 제작된 항체가 기존의 수법에 의해 제작된 항체와 동등한 혈중 농도 추이를 나타내는지 여부에 대해서 정상 마우스를 사용한 in vivo 시험에 의해 평가하였다. Whether the human IgG-type and mouse IgG-type antibodies produced by Fab Arm Exchange exhibited blood concentration trends equivalent to those produced by conventional methods was evaluated by an in vivo test using normal mice.

인간 IgG형 항체로서는 3종류의 항인간 글리피칸 3/항인간 CD3 이중특이성 항체：TR-G1drP1/N1, TR-G1dP17/N17, TR-G4dP1/N1을 인간 IgG형 Fab Arm Exchange에 의해 제작하였다. 또한 대조 항체로서는 가변영역은 상기와 동일하게 항인간 글리피칸 3/항인간 CD3를 갖고, 불변영역으로서는 MRAH-G1d(서열번호 3)의 EU 넘버링 118번째 Ala부터 시작되는 불변영역 G1d 및 MRAH-wtG4d(서열번호 5)의 EU 넘버링 118번째 Ala로부터 시작되는 불변영역 wtG4d에서 추가로 228번째 아미노산 잔기를 Ser에서 Pro로 치환하여 IgG1형의 힌지로 한 G4d에 대해 Knobs-into-Holes 개변(Nature, 1994, 372, 379-383)을 도입하여 제작한 이중특이성 항체 TR-G1dKiH, TR-G4dKiH를 사용하였다. 여기서 불변영역명 G1dKiH 및 G4dKiH란, 불변영역 G1d 또는 G4d에 대해 Knob 개변(349번째 아미노산 잔기를 Cys로, 366번째 아미노산 잔기를 Trp로 치환하는 개변)을 도입한 Knob쇄와, G1d 또는 G4d에 대해 Hole 개변(356번째 아미노산 잔기를 Cys로, 366번째 아미노산 잔기를 Ser로, 368번째 아미노산 잔기를 Ala로, 407번째 아미노산 잔기를 Val로 치환하는 개변)을 도입한 Hole쇄를 사용하여, 각각의 Knob쇄와 Hole쇄의 조합으로 발현된 불변영역을 나타낸다. As human IgG-type antibodies, three types of anti-human glypican 3/anti-human CD3 bispecific antibodies: TR-G1drP1/N1, TR-G1dP17/N17, and TR-G4dP1/N1 were prepared by human IgG-type Fab Arm Exchange. In addition, as a control antibody, the variable region has anti-human glypican 3/anti-human CD3 as described above, and as the constant region, the constant regions G1d and MRAH-wtG4d starting from EU numbering 118th Ala of MRAH-G1d (SEQ ID NO: 3). In the constant region wtG4d starting from EU numbering 118th Ala of (SEQ ID NO: 5), Knobs-into-Holes modification (Nature, 1994) , 372, 379-383) were used to introduce bispecific antibodies TR-G1dKiH, TR-G4dKiH. Herein, the constant region names G1dKiH and G4dKiH are a Knob chain in which Knob modifications (modification by replacing the 349th amino acid residue with Cys and the 366th amino acid residue with Trp) are introduced with respect to the constant regions G1d or G4d, and for G1d or G4d Using the Hole chain introduced with Hole modifications (modifications in which the 356th amino acid residue is replaced with Cys, the 366th amino acid residue is replaced with Ser, the 368th amino acid residue is replaced with Ala, and the 407th amino acid residue is replaced with Val), each Knob It represents the constant region expressed by the combination of the chain and the hole chain.

한편 마우스 IgG형 항체로서는 WO2009/125825에 기재되어 있는 항인간 IL-6 수용체 항체의 H쇄 가변영역인 H237의 서열을 갖고, 불변영역은 천연형 mIgG1의 서열인 H237-mIgG1(서열번호 73)에 대해, Fab-Arm Exchange용 개변을 도입한 H237-mIgG1mP3(서열번호 74), H237-mIgG1mN3(서열번호 75), H237-mIgG1mP4(서열번호 76), H237-mIgG1mN4(서열번호 77)를 제작하였다. 항체 L쇄로서는 항인간 IL-6 수용체 L쇄 가변영역인 L104의 서열을 사용하고, 이와 마우스 κ쇄의 불변영역인 mk1으로 이루어지는 L104-mk1(서열번호：78)을 사용하였다. 참고예 1의 방법에 따라 발현하여, H237-mIgG1mP3/L104-mk1, H237-mIgG1mN3/L104-mk1, H237-mIgG1mP4/L104-mk1, H237-mIgG1mN4/L104-mk1을 얻었다. 얻어진 호모체를 사용해서 Fab Arm Exchange를 행하여 SA-mIgG1mP3/mN3(H237-mIgG1mP3/L104-mk1과 H237-mIgG1mN3/L104-mk1의 조합), SA-mIgG1mP4/mN4(H237-mIgG1mP4/L104-mk1과 H237-mIgG1mN4/L104-mk1의 조합)를 얻었다. 또한 대조 항체로서는 H237-mIgG1과 L104-mk1을 사용하여 발현한 SA-mIgG1을 사용하였다. On the other hand, as a mouse IgG-type antibody, it has the sequence of H237 which is the H chain variable region of the anti-human IL-6 receptor antibody described in WO2009/125825, and the constant region is H237-mIgG1 (SEQ ID NO: 73) which is the sequence of native mIgG1. For Fab-Arm Exchange, H237-mIgG1mP3 (SEQ ID NO: 74), H237-mIgG1mN3 (SEQ ID NO: 75), H237-mIgG1mP4 (SEQ ID NO: 76), H237-mIgG1mN4 (SEQ ID NO: 77) were prepared. As the antibody L chain, the sequence of L104 which is an anti-human IL-6 receptor L chain variable region was used, and L104-mk1 (SEQ ID NO: 78) which consists of mk1 which is a constant region of a mouse ? chain was used. Expression was performed according to the method of Reference Example 1 to obtain H237-mIgG1mP3/L104-mk1, H237-mIgG1mN3/L104-mk1, H237-mIgG1mP4/L104-mk1, and H237-mIgG1mN4/L104-mk1. Fab arm exchange was performed using the obtained homo body, and SA-mIgG1mP3/mN3 (a combination of H237-mIgG1mP3/L104-mk1 and H237-mIgG1mN3/L104-mk1), SA-mIgG1mP4/mN4 (H237-mIgG1mk1/mP4/L104-mk1) H237-mIgG1mN4/L104-mk1) was obtained. In addition, SA-mIgG1 expressed using H237-mIgG1 and L104-mk1 were used as control antibodies.

Fab Arm Exchange는 모두 아래의 반응 조건에서 행하고, 반응 후 PBS에 투석하여 in vivo 시험에 사용하였다.Fab arm exchange was all carried out under the following reaction conditions, and after the reaction was dialyzed against PBS, it was used for the in vivo test.

반응 조건：TBS(Takara, pH 7.6) 중 [각 mAb] = 0.225 ㎎/㎖, [2-MEA(Sigma)] = 25 mM, 37℃, 17시간. Reaction conditions: [each mAb] = 0.225 mg/ml, [2-MEA (Sigma)] = 25 mM, 37 °C, 17 hours in TBS (Takara, pH 7.6).

정상 마우스(C57BL/6J mouse, Charles River Japan)에 인간 IgG형 항인간 글리피칸 3/항인간 CD3 이중특이성 항체(TR-G1dKiH, TR-G1drP1/N1, TR-G1dP17/N17, TR-G4dKiH, TR-G4dP1/N1), 항인간 IL-6 수용체 마우스 항체(SA-mIgG1, SA-mIgG1mP3/mN3 및 SA-mIgG1mP4/mN4)를 투여한 후의 각 항체의 체내동태를 평가하였다. 항체는 0.1 ㎎/mL로 조제된 것을 10 mL/㎏으로 꼬리정맥에 투여하였다. 항체 투여 후 5분, 2시간, 1일, 2일, 3일, 7일, 14일, 21일, 28일이 경과한 후에 당해 마우스로부터 채혈이 행해졌다. 채취된 혈액을 바로 4℃, 15,000 rpm으로 15분간 원심 분리함으로써 혈장이 얻어졌다. 분리한 혈장은 측정을 실시할 때까지 -20℃ 이하로 설정된 냉동고에 보존되었다.Human IgG-type anti-human glypican 3/anti-human CD3 bispecific antibodies (TR-G1dKiH, TR-G1drP1/N1, TR-G1dP17/N17, TR-G4dKiH, TR) in normal mice (C57BL/6J mouse, Charles River Japan) -G4dP1/N1) and anti-human IL-6 receptor mouse antibodies (SA-mIgG1, SA-mIgG1mP3/mN3 and SA-mIgG1mP4/mN4) were evaluated for in vivo kinetics of each antibody. The antibody prepared at 0.1 mg/mL was administered to the tail vein at 10 mL/kg. After 5 minutes, 2 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after antibody administration, blood was collected from the mice. Plasma was obtained by immediately centrifuging the collected blood at 4°C and 15,000 rpm for 15 minutes. The separated plasma was stored in a freezer set at -20°C or lower until measurement was performed.

(11-2) ECLIA법에 의한 혈장 중 이중특이성 항체 농도의 측정(11-2) Measurement of plasma bispecific antibody concentration by ECLIA method

마우스 혈장 중의 이중특이성 항체 농도는 ECLIA법으로 측정되었다. 먼저 가용형 인간 글리피칸 3를 MULTI-ARRAY 96well Plate(Meso Scale Discovery)에 분주하고, 4℃에서 하룻밤 정치함으로써 가용형 인간 글리피칸 3 고상화 플레이트가 제작되었다. 혈장 중 농도로서 200, 100, 50, 25, 12.5, 6.25, 3.125 ng/mL의 이중특이성 항체를 포함하는 검량선 시료와 100배 이상 희석된 마우스 혈장 측정시료가 조제되었다. 이들 검량선 시료 및 혈장 측정시료 100 μL가 각 웰에 분주된 가용형 인간 글리피칸 3 고상화 플레이트를 실온에서 2시간 교반시켰다. 계속해서 항인간 CD3 항체에 대한 토끼형 이디오타입 항체를 실온에서 1시간 교반시켰다. 그 후, Anti-Rabbit IgG-Sulfotag antibody(Meso Scale Discovery)를 실온에서 1시간 반응시키고, Read Buffer T(Meso Scale Discovery)를 첨가 후의 발광이 SECTOR Imager 2400(Meso Scale Discovery)으로 측정되었다. 마우스 혈장 중의 항체 농도는 검량선의 발광 시그널로부터 해석 소프트웨어 SOFTmax PRO(Molecular Devices)를 사용하여 산출되었다. 그 결과를 도 11에 나타내었다. 또한 PK 파라미터를 표 11에 나타낸다. 도 11 및 표 11에 나타내어진 결과로부터, 인간 IgG형 Fab Arm Exchange에 의해 제작된 이중특이성 항체는 모두 기존의 이중특이성 항체 제작기술인 Knobs-into-Holes 기술을 사용하여 제작된 대조 항체와 동등한 혈중 농도 추이를 나타내는 것이 명확해졌다. The concentration of the bispecific antibody in mouse plasma was measured by the ECLIA method. First, soluble human glypican 3 was dispensed on a MULTI-ARRAY 96 well plate (Meso Scale Discovery), and left overnight at 4° C. to prepare a soluble human glypican 3 solidified plate. A calibration curve sample containing 200, 100, 50, 25, 12.5, 6.25, 3.125 ng/mL of the bispecific antibody as a plasma concentration and a mouse plasma measurement sample diluted 100-fold or more were prepared. The soluble human glypican 3 solidified plate in which 100 µL of these calibration curve samples and plasma measurement samples were dispensed into each well was stirred at room temperature for 2 hours. Subsequently, the rabbit idiotype antibody against the anti-human CD3 antibody was stirred at room temperature for 1 hour. Then, Anti-Rabbit IgG-Sulfotag antibody (Meso Scale Discovery) was reacted at room temperature for 1 hour, and luminescence after addition of Read Buffer T (Meso Scale Discovery) was measured with SECTOR Imager 2400 (Meso Scale Discovery). The antibody concentration in mouse plasma was calculated using the analysis software SOFTmax PRO (Molecular Devices) from the luminescence signal of the calibration curve. The results are shown in FIG. 11 . In addition, the PK parameters are shown in Table 11. From the results shown in Figure 11 and Table 11, all of the bispecific antibodies produced by the human IgG-type Fab Arm Exchange had the same blood concentration as the control antibody produced using the Knobs-into-Holes technology, which is a conventional bispecific antibody production technology. It became clear to indicate the trend.

Figure 112016038698342-pct00011
Figure 112016038698342-pct00011

(11-3) ELISA법에 의한 혈장 중 항인간 IL-6 수용체 마우스 항체 농도의 측정(11-3) Measurement of anti-human IL-6 receptor mouse antibody concentration in plasma by ELISA method

마우스 혈장 중의 항인간 IL-6 수용체 마우스 항체 농도는 ELISA법으로 측정되었다. 먼저 가용형 인간 IL-6 수용체를 Nunc-Immuno Plate, MaxiSoup(Nalge nunc International)에 분주하고, 4℃에서 하룻밤 정치함으로써 가용형 인간 IL-6 수용체 고상화 플레이트가 제작되었다. 혈장 중 농도로서 2.50, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039 ㎍/mL의 항인간 IL-6 수용체 마우스 항체를 포함하는 검량선 시료와 100배 이상 희석된 마우스 혈장 측정시료가 조제되었다. 이들 검량선 시료 및 혈장 측정시료 100 μL가 각 웰에 분주된 가용형 인간 IL-6 수용체 고상화 플레이트를 실온에서 2시간 교반시켰다. 그 후 Anti-Mouse IgG-Peroxidase antibody(SIGMA)를 실온에서 2시간 반응시킨 반응액의 발색반응이 TMB One Component HRP Microwell Substrate(BioFX Laboratories)를 기질로서 사용해서 행해졌다. 1N-Sulfuric acid(Showa Chemical)를 첨가함으로써 반응이 정지된 각 웰의 반응액의 450 nm의 흡광도가 마이크로플레이트 리더로 측정되었다. 마우스 혈장 중의 항체 농도는 검량선의 흡광도로부터 해석 소프트웨어 SOFTmax PRO(Molecular Devices)를 사용하여 산출되었다. 그 결과를 도 12에 나타내고, 그의 항체 파라미터를 표 12에 나타낸다. 도 12 및 표 12에 나타내어진 결과로부터, 마우스 IgG형 Fab Arm Exchange로 제작된 항체는 천연형 mIgG1의 서열을 갖는 대조 항체와 동등한 혈중 농도 추이를 나타내는 것이 명확해졌다. The concentration of anti-human IL-6 receptor mouse antibody in mouse plasma was measured by ELISA method. First, a soluble human IL-6 receptor was dispensed on a Nunc-Immuno Plate, MaxiSoup (Nalge nunc International), and left overnight at 4° C. to prepare a soluble human IL-6 receptor immobilized plate. A calibration curve sample containing anti-human IL-6 receptor mouse antibody at concentrations of 2.50, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 µg/mL in plasma and a sample for measuring mouse plasma diluted 100-fold or more were prepared. The soluble human IL-6 receptor immobilized plate in which 100 µL of these calibration curve samples and plasma measurement samples were dispensed into each well was stirred at room temperature for 2 hours. After that, the reaction solution was reacted with Anti-Mouse IgG-Peroxidase antibody (SIGMA) at room temperature for 2 hours, followed by color development using TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a substrate. The absorbance at 450 nm of the reaction solution in each well where the reaction was stopped by adding 1N-sulfuric acid (Showa Chemical) was measured with a microplate reader. The antibody concentration in mouse plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices). The result is shown in FIG. 12, and the antibody parameter is shown in Table 12. From the results shown in Fig. 12 and Table 12, it was clarified that the antibody prepared with the mouse IgG-type Fab Arm Exchange exhibited a blood concentration trend equivalent to that of the control antibody having the native mIgG1 sequence.

Figure 112016038698342-pct00012
Figure 112016038698342-pct00012

〔참고예 1〕항체의 발현 벡터의 제작 및 항체의 발현과 정제[Reference Example 1] Preparation of antibody expression vector and antibody expression and purification

항체의 H쇄 및 L쇄의 염기서열을 코드하는 전장의 유전자의 합성은 Assemble PCR 등을 사용하여 당업자 공지의 방법으로 제작하였다. 아미노산 치환의 도입은 PCR 등을 사용하여 당업자 공지의 방법으로 행하였다. 얻어진 플라스미드 단편을 동물세포 발현 벡터에 삽입하여 H쇄 발현 벡터 및 L쇄 발현 벡터를 제작하였다. 얻어진 발현 벡터의 염기서열은 당업자 공지의 방법으로 결정하였다. 제작한 플라스미드를 인간 태아 신장암 세포 유래 HEK293H주(Invitrogen사) 또는 FreeStyle293 세포(Invitrogen사)에 일과성으로 도입하여 항체의 발현을 행하였다. 얻어진 배양상청을 회수한 후, 0.22 ㎛ 필터 MILLEX(R)-GV(Millipore) 또는 0.45 ㎛ 필터 MILLEX(R)-GV(Millipore)를 통과시켜 배양상청을 얻었다. 얻어진 배양상청으로부터 rProtein A Sepharose Fast Flow(GE 헬스케어) 또는 Protein G Sepharose 4 Fast Flow(GE 헬스케어)를 사용하여 당업자 공지의 방법으로 항체를 정제하였다. 정제 항체 농도는 분광광도계를 사용하여 280 nm에서의 흡광도를 측정하고, 얻어진 값으로부터 PACE 등의 방법에 의해 산출된 흡광계수를 사용하여 항체 농도를 산출하였다(Protein Science 1995 ; 4 : 2411-2423). The synthesis of the full-length gene encoding the nucleotide sequences of the H chain and L chain of the antibody was prepared by a method known to those skilled in the art using assemble PCR or the like. The amino acid substitution was introduced by a method known to those skilled in the art using PCR or the like. The obtained plasmid fragment was inserted into an animal cell expression vector to prepare an H chain expression vector and an L chain expression vector. The nucleotide sequence of the obtained expression vector was determined by a method known to those skilled in the art. The prepared plasmid was transiently introduced into HEK293H strain (Invitrogen) or FreeStyle293 cells (Invitrogen) derived from human fetal kidney cancer cells to perform antibody expression. After recovering the obtained culture supernatant, a 0.22 µm filter MILLEX(R)-GV (Millipore) or a 0.45 µm filter MILLEX(R)-GV (Millipore) was passed to obtain a culture supernatant. From the obtained culture supernatant, the antibody was purified by a method known to those skilled in the art using rProtein A Sepharose Fast Flow (GE Healthcare) or Protein G Sepharose 4 Fast Flow (GE Healthcare). The purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer, and from the obtained value, the antibody concentration was calculated using the extinction coefficient calculated by the method such as PACE (Protein Science 1995; 4: 2411-2423). .

〔참고예 2〕이온 교환 크로마토그래피에 의한 이중특이성 항체 생성률의 평가[Reference Example 2] Evaluation of bispecific antibody production rate by ion exchange chromatography

Priminence UFLC(시마즈 제작소)를 사용한 이온 교환 크로마토그래피 정제법에 있어서, 각 검체의 분리가 평가되었다. 이동상에는 25 mM MES 완충액, pH 5.0 및 500 mM의 염화나트륨을 포함하는 25 mM MES 완충액, pH 5.0를, 칼럼은 ProPac WCX-10(Thermo Scientific)을 사용하여 2액 혼합 그라디언트법에 의해 이중특이성 항체가 분리되었다. 데이터의 취득은 215 nm의 파장에서 실시하고, Empower2(Waters)를 사용하여 용출결과가 평가되었다. In the ion exchange chromatography purification method using Priminence UFLC (Shimadzu Corporation), the separation of each sample was evaluated. The bispecific antibody was prepared by the two-component mixed gradient method using 25 mM MES buffer, pH 5.0 and 25 mM MES buffer containing 500 mM sodium chloride, pH 5.0 for the mobile phase, and ProPac WCX-10 (Thermo Scientific) for the column. has been separated Data acquisition was performed at a wavelength of 215 nm, and the dissolution result was evaluated using Empower2 (Waters).

이중특이성 항체 생성률(%)은 이중특이성 항체의 면적값을 시스템 중에 존재하는 전 항체의 면적값으로 나누고, 100을 곱한 값을 사용하였다. 한쪽 호모체의 회수율이 나쁜 경우에는, 다른 한쪽 호모체 면적의 2배값을 이중특이성 항체의 면적값과 합친 값을 전 항체의 면적값으로서 계산하였다.The bispecific antibody production rate (%) was obtained by dividing the area value of the bispecific antibody by the area value of all antibodies present in the system, and multiplying by 100 was used. When the recovery rate of one homo body was poor, the value obtained by adding the double value of the area of the other homo body to the area value of the bispecific antibody was calculated as the area value of all antibodies.

〔참고예 3〕Tm의 측정[Reference Example 3] Measurement of Tm

CH3 도메인의 Tm의 측정은 Rotor-gene Q(QIAGEN)를 사용하여 당업자 공지의 방법으로 행하였다. 항체 농도 0.1 ㎎/mL, SYPRO orange 농도 10X concentrate로 혼합한 샘플을 30도에서 99도까지 승온하여 형광강도(여기파장 470 nm, 형광파장 555 nm)를 0.4도마다 측정하였다. 측정은 PBS(Sigma, pH 7.4) 중에서 행하였다. 해석은 Rotor-gene Q series software를 사용하여 행하고, 형광강도의 1차 미분에 의해 구한 변곡점을 Tm으로 하였다. MRAH의 Tm은 CH2가 70℃ 부근, Fab가 95℃ 부근, H54의 Tm은 CH2가 70℃ 부근, Fab가 90℃ 부근인 것을 이용하여 CH3 도메인의 Tm이 산출되었다.Measurement of the Tm of the CH3 domain was performed by a method known to those skilled in the art using Rotor-gene Q (QIAGEN). The fluorescence intensity (excitation wavelength 470 nm, fluorescence wavelength 555 nm) was measured every 0.4 degrees by heating the sample mixed with antibody concentration 0.1 mg/mL and SYPRO orange concentration 10X concentrate from 30 degrees to 99 degrees. Measurement was performed in PBS (Sigma, pH 7.4). Analysis was performed using Rotor-gene Q series software, and the inflection point obtained by the first derivative of fluorescence intensity was used as Tm. The Tm of the CH3 domain was calculated using that the Tm of MRAH was around 70°C for CH2, around 95°C for Fab, the Tm for H54 was around 70°C for CH2, and around 90°C for Fab.

〔참고예 4〕SPR에 의한 상호작용 해석[Reference Example 4] Interaction analysis by SPR

(4-1) FcγR의 조제방법 및 개변 항체와 FcγR의 상호작용 해석방법(4-1) Method for preparing FcγR and analysis method for interaction between modified antibody and FcγR

FcγR의 세포외 도메인을 아래의 방법으로 조제하였다. 먼저 FcγR의 세포외 도메인의 유전자의 합성을 당업자 공지의 방법으로 실시하였다. 이때 각 FcγR의 서열은 NCBI에 등록되어 있는 정보를 토대로 제작하였다. 구체적으로는, FcγRI에 대해서는 NCBI의 허가번호 NM_000566.3의 서열, FcγRIIa에 대해서는 NCBI의 허가번호 NM_001136219.1의 서열, FcγRIIb에 대해서는 NCBI의 허가번호 NM_004001.3의 서열, FcγRIIIa에 대해서는 NCBI의 허가번호 NM_001127593.1의 서열, FcγRIIIb에 대해서는 NCBI의 허가번호 NM_000570.3의 서열을 토대로 제작하고, C말단에 His 태그를 부여하였다. 또한 FcγRIIa, FcγRIIIa, FcγRIIIb에 대해서는 다형이 알려져 있는데, 다형 부위에 대해서는 FcγRIIa에 대해서는 J. Exp. Med., 1990, 172: 19-25, FcγRIIIa에 대해서는 J. Clin. Invest., 1997, 100 (5): 1059-1070, FcγRIIIb에 대해서는 J. Clin. Invest., 1989, 84, 1688-1691을 참고로 하여 제작하였다. The extracellular domain of FcγR was prepared by the following method. First, the synthesis of the gene of the extracellular domain of FcγR was performed by a method known to those skilled in the art. At this time, the sequence of each FcγR was prepared based on the information registered in NCBI. Specifically, the sequence of NCBI permission number NM_000566.3 for FcγRI, the sequence of NCBI permission number NM_001136219.1 for FcγRIIa, the sequence of NCBI permission number NM_004001.3 for FcγRIIb, and the NCBI permission number for FcγRIIIa The sequence of NM_001127593.1 and FcγRIIIb were prepared based on the sequence of NCBI permission number NM_000570.3, and a His tag was attached to the C-terminus. In addition, polymorphisms are known for FcγRIIa, FcγRIIIa, and FcγRIIIb. For FcγRIIa, polymorphism sites are described in J. Exp. Med., 1990, 172: 19-25, J. Clin for FcγRIIIa. Invest., 1997, 100 (5): 1059-1070, J. Clin for FcγRIIIb. Invest., 1989, 84, was prepared with reference to 1688-1691.

얻어진 유전자 단편을 동물세포 발현 벡터에 삽입하여 발현 벡터를 제작하였다. 제작한 발현 벡터를 인간 태아 신장암 세포 유래 FreeStyle293세포(Invitrogen사)에 일과성으로 도입하여 목적 단백질을 발현시켰다. 배양하고 얻어진 배양상청을 회수한 후, 0.22 ㎛ 필터를 통과시켜 배양상청을 얻었다. 얻어진 배양상청은 원칙에 따라 다음의 4스텝으로 정제하였다. 제1 스텝은 양이온 교환 칼럼크로마토그래피(SP Sepharose FF), 제2 스텝은 His 태그에 대한 친화성 칼럼크로마토그래피(HisTrap HP), 제3 스텝은 겔 여과 칼럼크로마토그래피(Superdex200), 제4 스텝은 무균 여과를 실시하였다. 단, FcγRI에 대해서는 제1 스텝에 Q sepharose FF를 사용한 음이온 교환 칼럼크로마토그래피를 실시하였다. 정제한 단백질에 대해서는 분광광도계를 사용하여 280 nm에서의 흡광도를 측정하고, 얻어진 값으로부터 PACE 등의 방법에 의해 산출된 흡광계수를 사용하여 정제 단백질의 농도를 산출하였다(Protein Science 1995 ; 4 : 2411-2423). The obtained gene fragment was inserted into an animal cell expression vector to prepare an expression vector. The prepared expression vector was transiently introduced into FreeStyle293 cells (Invitrogen) derived from human fetal kidney cancer cells to express the target protein. After culturing and collecting the obtained culture supernatant, it was passed through a 0.22 μm filter to obtain a culture supernatant. The obtained culture supernatant was purified in the following 4 steps according to the principle. The first step is cation exchange column chromatography (SP Sepharose FF), the second step is affinity column chromatography for His tag (HisTrap HP), the third step is gel filtration column chromatography (Superdex200), and the fourth step is Aseptic filtration was performed. However, for FcγRI, anion exchange column chromatography using Q sepharose FF was performed in the first step. For the purified protein, the absorbance at 280 nm was measured using a spectrophotometer, and the concentration of the purified protein was calculated using the extinction coefficient calculated by the method such as PACE from the obtained value (Protein Science 1995; 4: 2411). -2423).

Biacore T100(GE 헬스케어), Biacore T200(GE 헬스케어), Biacore A100, Biacore 4000을 사용하여 각 개변 항체와 상기에서 조제한 Fcγ 수용체의 상호작용 해석을 행하였다. 러닝 버퍼에는 HBS-EP+(GE 헬스케어)를 사용하고, 측정 온도는 25℃로 하였다. Series S Sensor Chip CM5(GE 헬스케어) 또는 Series S sensor Chip CM4(GE 헬스케어)에 아민 커플링법에 의해 항원 펩티드, Protein A(Thermo Scientific), Protein A/G(Thermo Scientific), Protein L(ACTIGEN 또는 BioVision)을 고정화한 칩 또는 Series S Sensor Chip SA(certified)(GE 헬스케어)에 대해 사전에 비오틴화해 둔 항원 펩티드를 상호작용시켜 고정화한 칩을 사용하였다. Biacore T100 (GE Healthcare), Biacore T200 (GE Healthcare), Biacore A100, and Biacore 4000 were used to analyze the interaction between each modified antibody and the Fcγ receptor prepared above. HBS-EP+ (GE Healthcare) was used as the running buffer, and the measurement temperature was set to 25°C. Antigen peptide, Protein A (Thermo Scientific), Protein A/G (Thermo Scientific), Protein L (ACTIGEN) by amine coupling to Series S Sensor Chip CM5 (GE Healthcare) or Series S sensor Chip CM4 (GE Healthcare) Or BioVision) immobilized chip or Series S Sensor Chip SA (certified) (GE Healthcare) was used to interact with and immobilized antigen peptides in advance.

이들 센서 칩에 목적의 항체를 챕쳐시키고, 러닝 버퍼로 희석한 Fcγ 수용체를 상호작용시켜 항체에 대한 결합량을 측정하고 항체 간에 비교하였다. 단, Fcγ 수용체의 결합량은 캡쳐한 항체의 양에 의존하기 때문에 각 항체의 캡쳐량으로 Fcγ 수용체의 결합량을 나눈 보정값으로 비교하였다. 또한 10 mM glycine-HCl, pH 1.5를 반응시킴으로써 센서 칩에 캡쳐한 항체를 세정하고, 센서 칩을 재생하여 반복해서 사용하였다. The target antibody was captured in these sensor chips, and the Fcγ receptor diluted with a running buffer was interacted to measure the amount of binding to the antibody and compared between the antibodies. However, since the amount of Fcγ receptor binding depends on the amount of the captured antibody, it was compared with a correction value obtained by dividing the amount of binding of the Fcγ receptor by the capture amount of each antibody. In addition, the antibody captured on the sensor chip was washed by reacting with 10 mM glycine-HCl, pH 1.5, and the sensor chip was regenerated and used repeatedly.

또한 각 개변 항체의 FcγR에 대한 KD값을 산출하기 위해 속도론적인 해석은 아래의 방법으로 실시하였다. 먼저 상기 센서 칩에 목적의 항체를 캡쳐시키고 러닝 버퍼로 희석한 Fcγ 수용체를 상호작용시켜, 얻어진 센서그램에 대해 Biacore Evaluation Software에 의해 측정결과를 1:1 Langmuir binding model로 global fitting시킴으로써 결합속도상수 ka(L/mol/s), 해리속도상수 kd(1/s)를 산출하고, 그 값으로부터 해리상수 KD(mol/L)를 산출하였다. In addition, kinetic analysis was performed in the following way to calculate the KD value for FcγR of each modified antibody. First, the target antibody was captured on the sensor chip, and the Fcγ receptor diluted with a running buffer was interacted with, and the binding rate constant ka was performed by global fitting of the measurement result to the 1:1 Langmuir binding model by Biacore Evaluation Software for the obtained sensorgram. (L/mol/s), the dissociation rate constant kd (1/s) was calculated, and the dissociation constant KD (mol/L) was calculated from the value.

(4-2) FcRn의 조제방법 및 개변 항체와 FcRn의 상호작용 해석방법(4-2) Method for preparing FcRn and method for analyzing the interaction between modified antibody and FcRn

FcRn은 FcRn과 β2-마이크로글로불린의 복합체이다. 공표되어 있는 인간 FcRn 유전자 서열을 토대로 올리고 DNA 프라이머를 조제하였다(J Exp Med. 1994 Dec 1; 180(6): 2377-81). 유전자 전체를 코드하는 DNA 단편은 조제한 프라이머 및 주형으로서 인간 cDNA(Human Placenta Marathon-Ready cDNA, Clontech)를 사용하여 PCR에 의해 조제하였다. 얻어진 DNA 단편을 주형으로서 사용하고, 시그널영역(Met1~Leu290)을 함유하는 세포외 도메인을 코드하는 DNA 단편을 PCR에 의해 증폭하여 포유동물 세포 발현 벡터에 삽입하였다. 마찬가지로 공표되어 있는 인간 β2-마이크로글로불린 유전자 서열(Proc. Natl. Acad. Sci. U.S.A. 99 (26): 16899-16903 (2002))을 토대로 올리고 DNA 프라이머를 조제하였다. 유전자 전체를 코드하는 DNA 단편은 조제한 프라이머 및 주형으로서 인간 cDNA(Human Placenta Marathon-Ready cDNA, Clontech)를 사용하여 PCR에 의해 조제하였다. 얻어진 DNA 단편을 주형으로서 사용하고, 시그널영역(Met1~Met119)을 함유하는 단백질 전체를 코드하는 DNA 단편을 PCR에 의해 증폭하여 포유동물 세포 발현 벡터에 삽입하였다. FcRn is a complex of FcRn and β2-microglobulin. Oligo DNA primers were prepared based on the published human FcRn gene sequence (J Exp Med. 1994 Dec 1; 180(6): 2377-81). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as the prepared primer and template. Using the obtained DNA fragment as a template, a DNA fragment encoding an extracellular domain containing a signal region (Met1-Leu290) was amplified by PCR and inserted into a mammalian cell expression vector. Similarly, oligo DNA primers were prepared based on the published human β2-microglobulin gene sequence (Proc. Natl. Acad. Sci. U.S.A. 99 (26): 16899-16903 (2002)). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as the prepared primer and template. Using the obtained DNA fragment as a template, a DNA fragment encoding the entire protein containing the signal region (Met1 to Met119) was amplified by PCR and inserted into a mammalian cell expression vector.

가용형 인간 FcRn을 아래의 순서로 발현시켰다. 인간 FcRn(서열번호：30) 및 β2-마이크로글로불린(서열번호：31)을 발현시키기 위해 구축한 플라스미드를 PEI(Polyscience)를 사용한 리포펙션법에 의해 인간 태아 신장암 유래 세포주 HEK293H(Invitrogen)의 세포에 도입하였다. 얻어진 배양상청을 회수하고, IgG Sepharose 6 Fast Flow(Amersham Biosciences)를 사용하여 정제한 후에 HiTrap Q HP(GE Healthcare)(J Immunol. 2002 Nov 1; 169(9): 5171-80)를 사용함으로써 FcRn을 추가로 정제하였다. Soluble human FcRn was expressed in the following order. A plasmid constructed to express human FcRn (SEQ ID NO: 30) and β2-microglobulin (SEQ ID NO: 31) was subjected to lipofection using PEI (Polyscience) cells of the human fetal kidney cancer-derived cell line HEK293H (Invitrogen). introduced into The obtained culture supernatant was recovered, purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences), and then FcRn by using HiTrap Q HP (GE Healthcare) (J Immunol. 2002 Nov 1; 169(9): 5171-80). was further purified.

Biacore를 사용한 항체와 FcRn의 상호작용을 평가하는 측정 시스템으로서, J Immunol. 2009;182(12):7663-71.에 기재된 센서 칩으로 항체를 고정화하여 인간 FcRn을 애널라이트로 하는 시스템이 보고되어 있다. 그 목적을 위해 인간 FcRn을 참고예 4에 기재된 바와 같이 조제하였다. 본 시스템을 사용하여 Fv4-IgG1, Fv4-IgG1-v1 및 Fv4-IgG1-v2의 pH 6.0 및 pH 7.4에 있어서의 인간 FcRn으로의 결합 활성(해리상수 KD)의 평가를 행하였다. 피험물질인 항체는 Series S 센서 칩 CM5에 직접 고정화하여 시험에 제공하였다. 센서 칩으로의 항체의 고정화는 러닝 버퍼로서 50 mmol/L 인산나트륨/150 mmol/L NaCl, 0.05%(v/v%) Surfactant P20 pH 6.0을 사용하고, 아민 커플링 키트를 사용하여 제조사의 매뉴얼에 따라 고정화량 500RU를 목표로 실시하였다. As a measurement system for evaluating the interaction of antibody and FcRn using Biacore, J Immunol. 2009;182(12):7663-71. has reported a system in which an antibody is immobilized with a sensor chip and human FcRn is used as an analyte. For that purpose, human FcRn was prepared as described in Reference Example 4. The binding activity (dissociation constant KD) of Fv4-IgG1, Fv4-IgG1-v1 and Fv4-IgG1-v2 to human FcRn at pH 6.0 and pH 7.4 was evaluated using this system. The antibody as a test substance was directly immobilized on the Series S sensor chip CM5 and used for testing. Immobilization of the antibody to the sensor chip was performed using 50 mmol/L sodium phosphate/150 mmol/L NaCl, 0.05% (v/v%) Surfactant P20 pH 6.0 as running buffer, and the manufacturer's manual using an amine coupling kit. Accordingly, the immobilization amount of 500RU was targeted.

러닝 버퍼로서 50 mmol/L 인산나트륨/150 mmol/L NaCl, 0.05% Surfactant P20 pH 6.0, 또는 50 mmol/L 인산나트륨/150 mmol/L NaCl, 0.05% Surfactant P20 pH 7.4를 사용하여 제작한 센서 칩을 사용해서 측정을 실시하였다. 측정은 모두 25℃에서 행하였다. 인간 FcRn 희석액과 러닝 버퍼(대조 용액으로서)를 유속 5 μL/min로 10분간 인젝트하고, 센서 칩 상의 항체에 인간 FcRn을 상호작용시켰다. 그 후 유속 5 μL/min로 1분간 러닝 버퍼를 흘리고 FcRn의 해리를 관찰한 후, 20 mmol/L Tris-HCl/150 mmol/L NaCl, pH 8.1을 유속 30 μL/min로 15초간 인젝트를 2회 반복해서 센서 칩을 재생하였다. Sensor chip fabricated using 50 mmol/L sodium phosphate/150 mmol/L NaCl, 0.05% Surfactant P20 pH 6.0, or 50 mmol/L sodium phosphate/150 mmol/L NaCl, 0.05% Surfactant P20 pH 7.4 as running buffer was used for measurement. All measurements were performed at 25°C. Human FcRn dilution and running buffer (as control solution) were injected at a flow rate of 5 µL/min for 10 minutes, and human FcRn was allowed to interact with the antibody on the sensor chip. After that, run the running buffer at a flow rate of 5 μL/min for 1 minute and observe the dissociation of FcRn, then inject 20 mmol/L Tris-HCl/150 mmol/L NaCl, pH 8.1 at a flow rate of 30 μL/min for 15 seconds. The sensor chip was regenerated twice repeatedly.

또한 각 개변 항체의 FcRn에 대한 KD값을 산출하기 위해 속도론적인 해석은 아래의 방법에 따라 실시하였다. 먼저, 상기 센서 칩에 목적의 항체를 캡쳐시키고 러닝 버퍼로 희석한 FcRn을 상호작용시켜, 얻어진 센서그램에 대해 Biacore Evaluation Software에 의해 측정결과를 1:1 Langmuir binding model로 global fitting시킴으로써 결합속도상수 ka(L/mol/s), 해리속도상수 kd(1/s)를 산출하고, 그 값으로부터 해리상수 KD(mol/L)를 산출하였다. In addition, kinetic analysis was performed according to the following method in order to calculate the KD value for FcRn of each modified antibody. First, by capturing the target antibody on the sensor chip and interacting with FcRn diluted with a running buffer, the binding rate constant ka is performed by global fitting of the measurement result to the 1:1 Langmuir binding model by Biacore Evaluation Software for the obtained sensorgram. (L/mol/s), the dissociation rate constant kd (1/s) was calculated, and the dissociation constant KD (mol/L) was calculated from the value.

(4-3) mFcγR의 조제방법 및 개변 항체와 mFcγR의 상호작용 해석방법(4-3) Method for preparing mFcγR and analysis method for interaction between modified antibody and mFcγR

마우스의 FcγR의 세포외 도메인을 아래의 방법으로 조제하였다. 먼저 FcγR의 세포외 도메인의 유전자의 합성을 당업자 공지의 방법으로 실시하였다. 이때, 각 FcγR의 서열은 NCBI에 등록되어 있는 정보를 토대로 제작하였다. 구체적으로는, mFcγRI에 대해서는 NCBI Reference Sequence: NP_034316.1, mFcγRII에 대해서는 NCBI Reference Sequence: NP_034317.1, mFcγRIII에 대해서는 NCBI Reference Sequence: NP_034318.2, mFcγRIV에 대해서는 NCBI Reference Sequence: NP_653142.2의 서열을 토대로 제작하고, C말단에 His 태그를 부가하였다. The extracellular domain of mouse FcγR was prepared by the following method. First, the synthesis of the gene of the extracellular domain of FcγR was performed by a method known to those skilled in the art. At this time, the sequence of each FcγR was prepared based on the information registered in NCBI. Specifically, the sequence of NCBI Reference Sequence: NP_034316.1 for mFcγRI, NCBI Reference Sequence: NP_034317.1 for mFcγRII, NCBI Reference Sequence: NP_034318.2 for mFcγRIII, and NCBI Reference Sequence: NP_653142.2 for mFcγRIV Based on the preparation, a His tag was added to the C terminus.

얻어진 유전자 단편을 동물세포 발현 벡터에 삽입하여 발현 벡터를 제작하였다. 제작한 발현 벡터를 인간 태아 신장암 세포 유래 FreeStyle293세포(Invitrogen사)에 일과성으로 도입하여 목적 단백질의 발현을 행하였다. 얻어진 배양상청을 회수한 후, 0.22 ㎛ 필터를 통과시켜 배양상청을 얻었다. 얻어진 배양상청은 원칙에 따라 다음의 4스텝으로 정제하였다. 제1 스텝은 이온 교환 칼럼크로마토그래피, 제2 스텝은 His 태그에 대한 친화성 칼럼크로마토그래피(HisTrap HP), 제3 스텝은 겔 여과 칼럼크로마토그래피(Superdex200), 제4 스텝은 무균 여과를 실시하였다. 제1 스텝의 이온 교환 칼럼크로마토그래피에 대해서 mFcγRI는 Q Sepharose HP, mFcγRII 및 mFcγRIV는 SP Sepharose FF, mFcγRIII는 SP Sepharose HP를 사용하였다. 또한 제3 스텝 이후에서 사용한 용매는 D-PBS(-)로 하였으나, mFcγRIII에 대해서는 0.1M Arginine을 포함하는 D-PBS(-)로 하였다. 정제한 단백질에 대해서는 분광광도계를 사용하여 280 nm에서의 흡광도를 측정하고, 얻어진 값으로부터 PACE 등의 방법에 의해 산출된 흡광계수를 사용하여 정제 단백질의 농도를 산출하였다(Protein Science 1995 ; 4 : 2411-2423). The obtained gene fragment was inserted into an animal cell expression vector to prepare an expression vector. The prepared expression vector was transiently introduced into FreeStyle293 cells derived from human fetal kidney cancer cells (Invitrogen) to express the target protein. After recovering the obtained culture supernatant, it was passed through a 0.22 μm filter to obtain a culture supernatant. The obtained culture supernatant was purified in the following 4 steps according to the principle. The first step was ion exchange column chromatography, the second step was affinity column chromatography for His tag (HisTrap HP), the third step was gel filtration column chromatography (Superdex200), and the fourth step was aseptic filtration. . For the ion exchange column chromatography of the first step, Q Sepharose HP for mFcγRI, SP Sepharose FF for mFcγRII and mFcγRIV, and SP Sepharose HP for mFcγRIII were used. In addition, the solvent used after the third step was D-PBS (-), but for mFcγRIII, D-PBS (-) containing 0.1 M Arginine was used. For the purified protein, the absorbance at 280 nm was measured using a spectrophotometer, and the concentration of the purified protein was calculated using the extinction coefficient calculated by the method such as PACE from the obtained value (Protein Science 1995; 4: 2411). -2423).

Biacore T100(GE 헬스케어), Biacore T200(GE 헬스케어), Biacore A100, Biacore 4000을 사용하여 각 개변 항체와 상기에서 조제한 Fcγ 수용체의 상호작용 해석을 행하였다. 러닝 버퍼에는 HBS-EP+(GE 헬스케어)를 사용하고, 측정 온도는 25℃로 하였다. Series S Sensor Chip CM5(GE 헬스케어) 또는 Series S sensor Chip CM4(GE 헬스케어)에 아민 커플링법에 의해 항원 펩티드, Protein A(Thermo Scientific), Protein A/G(Thermo Scientific), Protein L(ACTIGEN 또는 BioVision)을 고정화한 칩 또는 Series S Sensor Chip SA(certified)(GE 헬스케어)에 대해 사전에 비오틴화해 둔 항원 펩티드를 상호작용시켜 고정화한 칩을 사용하였다. Biacore T100 (GE Healthcare), Biacore T200 (GE Healthcare), Biacore A100, and Biacore 4000 were used to analyze the interaction between each modified antibody and the Fcγ receptor prepared above. HBS-EP+ (GE Healthcare) was used as the running buffer, and the measurement temperature was set to 25°C. Antigen peptide, Protein A (Thermo Scientific), Protein A/G (Thermo Scientific), Protein L (ACTIGEN) by amine coupling to Series S Sensor Chip CM5 (GE Healthcare) or Series S sensor Chip CM4 (GE Healthcare) Or BioVision) immobilized chip or Series S Sensor Chip SA (certified) (GE Healthcare) was used to interact with and immobilized antigen peptides in advance.

이들 센서 칩에 목적의 항체를 챕쳐시키고, 러닝 버퍼로 희석한 FcγR을 상호작용시켜 항체에 대한 결합량을 측정하고 항체 간에 비교하였다. 단, FcγR의 결합량은 캡쳐한 항체의 양에 의존하기 때문에 각 항체의 캡쳐량으로 FcγR의 결합량을 나눈 보정값으로 비교하였다. 또한 10 mM glycine-HCl, pH 1.5를 반응시킴으로써 센서 칩에 캡쳐한 항체를 세정하고, 센서 칩을 재생하여 반복해서 사용하였다. The target antibody was captured in these sensor chips, and FcγR diluted with a running buffer was interacted to measure the amount of binding to the antibody, and the antibodies were compared. However, since the binding amount of FcγR depends on the amount of the captured antibody, it was compared with a correction value obtained by dividing the binding amount of FcγR by the capture amount of each antibody. In addition, the antibody captured on the sensor chip was washed by reacting with 10 mM glycine-HCl, pH 1.5, and the sensor chip was regenerated and used repeatedly.

또한 각 개변 항체의 FcγR에 대한 KD값을 산출하기 위해 속도론적인 해석은 아래의 방법에 따라 실시하였다. 먼저 상기 센서 칩에 목적의 항체를 캡쳐시키고 러닝 버퍼로 희석한 FcγR을 상호작용시켜, 얻어진 센서그램에 대해 Biacore Evaluation Software에 의해 측정결과를 1:1 Langmuir binding model로 global fitting시킴으로써 결합속도상수 ka(L/mol/s), 해리속도상수 kd(1/s)를 산출하고, 그 값으로부터 해리상수 KD(mol/L)를 산출하였다. In addition, in order to calculate the KD value for FcγR of each modified antibody, kinetic analysis was performed according to the following method. First, the target antibody was captured on the sensor chip, and FcγR diluted with a running buffer was interacted, and the binding rate constant ka ( L/mol/s) and the dissociation rate constant kd (1/s) were calculated, and the dissociation constant KD (mol/L) was calculated from the values.

(4-4) mFcRn의 조제방법 및 개변 항체와 mFcRn의 상호작용 해석방법(4-4) Method for preparing mFcRn and analysis method for interaction between modified antibody and mFcRn

Biacore T100, Biacore T200, Biacore A100, Biacore 4000(GE Healthcare)을 사용하여 마우스 FcRn과 항체의 속도론적 해석을 행하였다. 센서 칩 CM4(GE Healthcare) 상에 아민 커플링법으로 프로테인 L(ACTIGEN)을 적당량 고정화하고, 거기에 목적의 항체를 포착시켰다. 다음으로 FcRn 희석액과 러닝 버퍼(대조 용액으로서)를 인젝트하고, 센서 칩 상에 포착시킨 항체에 마우스 FcRn을 상호작용시켰다. 러닝 버퍼에는 50 mmol/L 인산나트륨, 150 mmol/L NaCl, 0.05% (w/v) Tween20, pH 6.0을 사용하고, FcRn의 희석에도 각각의 버퍼를 사용하였다. 칩의 재생에는 10 mmol/L 글리신-HCl, pH 1.5를 사용하였다. 측정은 모두 25℃에서 실시하였다. 측정으로 얻어진 센서그램으로부터 키네틱스 파라미터인 결합속도상수 ka(1/Ms) 및 해리속도상수 kd(1/s)를 산출하고, 그 값을 토대로 각 항체의 마우스 FcRn에 대한 KD(M)를 산출하였다. 각 파라미터의 산출에는 Biacore Evaluation Software(GE Healthcare)를 사용하였다. Kinetic analysis of mouse FcRn and antibody was performed using Biacore T100, Biacore T200, Biacore A100, and Biacore 4000 (GE Healthcare). An appropriate amount of protein L (ACTIGEN) was immobilized on a sensor chip CM4 (GE Healthcare) by an amine coupling method, and the target antibody was captured there. Next, the FcRn dilution and running buffer (as a control solution) were injected, and mouse FcRn was made to interact with the antibody captured on the sensor chip. 50 mmol/L sodium phosphate, 150 mmol/L NaCl, 0.05% (w/v) Tween20, pH 6.0 were used as the running buffer, and each buffer was also used for the dilution of FcRn. For regeneration of the chip, 10 mmol/L glycine-HCl, pH 1.5 was used. All measurements were carried out at 25°C. From the sensorgram obtained by the measurement, the association rate constant ka (1/Ms) and the dissociation rate constant kd (1/s), which are kinetic parameters, are calculated, and the KD (M) for mouse FcRn of each antibody is calculated based on the values. did. Biacore Evaluation Software (GE Healthcare) was used to calculate each parameter.

〔참고예 5〕CE-IEF[Reference Example 5] CE-IEF

CE-IEF의 측정은 PA800 Plus(Beckman Coulter)를 사용하여 당업자 공지의 방법으로 행하였다. Pharmalyte는 broad range가 5-8과 8-10.5인 것을 등량 혼합하여 pI range 5-10.5에서 분석을 행하였다. 4 ㎎/mL의 항체용액을 사용하여 분석하고, 결과는 32 karat software(Beckman Coulter)를 사용하여 해석하였다. 이중특이성 항체 생성률(%)은 이중특이성 항체의 면적값을 시스템 중에 존재하는 전 항체의 면적값으로 나누고, 100을 곱한 값을 사용하였다. CE-IEF was measured by a method known to those skilled in the art using PA800 Plus (Beckman Coulter). Pharmalyte was analyzed in the pi range 5-10.5 by mixing equal amounts of those having a broad range of 5-8 and 8-10.5. Analysis was performed using an antibody solution of 4 mg/mL, and the results were analyzed using 32 karat software (Beckman Coulter). The bispecific antibody production rate (%) was obtained by dividing the area value of the bispecific antibody by the area value of all antibodies present in the system, and multiplying by 100 was used.

〔참고예 6〕세포상해활성의 측정[Reference Example 6] Measurement of cytotoxic activity

(6-1) 인간 말초혈 단핵구(PBMC)용액의 조제(6-1) Preparation of human peripheral blood mononuclear cell (PBMC) solution

1,000 단위/mL의 헤파린용액(노보·헤파린 주사 5천 단위，노보·노르디스크사)을 사전에 100 μL 주입한 주사기를 사용하여, 건강한 정상인 자원봉사자(성인)로부터 말초혈 50 mL가 채취되었다. PBS(-)로 2배 희석한 후에 4등분된 말초혈이 15 mL의 Ficoll-Paque PLUS를 사전에 주입하여 원심조작이 행해진 Leucosep 림프구 분리관(Cat. No. 227290, Greiner bio-one사)에 첨가되었다. 당해 분리관의 원심분리(2,150 rpm, 10분간, 실온) 후 단핵구 분획층이 분취되었다. 10% FBS를 포함하는 Dulbecco's Modified Eagle's Medium(SIGMA사, 이하 10% FBS/D-MEM)로 1회 단핵구 분획의 세포가 세정된 후, 당해 세포는 10% FBS/D-MEM을 사용하여 그 세포밀도가 4×106 cells/mL로 조제되었다. 이와 같이 조제된 세포용액이 인간 PBMC용액으로서 이후의 시험에 사용되었다. Using a syringe in which 100 µL of a 1,000 units/mL heparin solution (5,000 units of Novo/Heparin injection, Novo Nordisk) was previously injected, 50 mL of peripheral blood was collected from healthy volunteers (adults). After two-fold dilution with PBS(-), 15 mL of Ficoll-Paque PLUS was pre-injected into quartered peripheral blood, and centrifuged into a Leucosep lymphocyte separation tube (Cat. No. 227290, Greiner bio-one). was added After centrifugation of the separation tube (2,150 rpm, 10 minutes, room temperature), the mononuclear fraction layer was collected. After washing the cells of the mononuclear fraction once with Dulbecco's Modified Eagle's Medium (SIGMA, hereinafter, 10% FBS/D-MEM) containing 10% FBS, the cells were washed with 10% FBS/D-MEM. The density was adjusted to 4×10 6 cells/mL. The cell solution thus prepared was used as a human PBMC solution for subsequent tests.

(6-2) 세포상해활성의 측정(6-2) Measurement of cytotoxic activity

세포상해활성은 xCELLigence 실시간 셀 애널라이저(료슈 다이아그노스틱스사)를 사용한 세포 증식 억제율로 평가되었다. 표적 세포에는 SK-HEP-1 세포주에 인간 GPC3를 강제 발현시켜 수립한 SK-pca13a 세포주가 사용되었다. SK-pca13a를 접시로부터 박리하여 1×104 cells/well이 되도록 E-Plate 96(료슈 다이아그노스틱스사) 플레이트에 100 μL/well로 뿌리고, xCELLigence 실시간 셀 애널라이저를 사용하여 생세포의 측정이 개시되었다. 다음날 xCELLigence 실시간 셀 애널라이저로부터 플레이트를 꺼내, 당해 플레이트에 각 농도(0.004, 0.04, 0.4, 4 ㎍/㎖)로 조제한 각 항체 50 μL가 첨가되었다. 실온에서 15분간 반응시킨 후에 (6-1)에서 조제된 인간 PBMC용액 50 μL(2×104 cells/well)가 첨가되고, xCELLigence 실시간 셀 애널라이저에 당해 플레이트를 재세팅함으로써 생세포의 측정이 개시되었다. 반응은 5% 탄산가스, 37℃ 조건하에서 행해지고, 인간 PBMC 첨가 72시간 후의 Cell Index값으로부터, 하기 식에 의해 세포 증식 억제율(%)이 구해졌다. 또한 계산에 사용한 Cell Index값은 항체 첨가 직전의 Cell Index값이 1이 되도록 정규화한 후의 수치가 사용되었다. The cytotoxic activity was evaluated by the cell proliferation inhibition rate using the xCELLigence real-time cell analyzer (Ryoshu Diagnostics). As the target cells, the SK-pca13a cell line established by forced expression of human GPC3 in the SK-HEP-1 cell line was used. SK-pca13a was peeled off the plate and sown at 100 μL/well on an E-Plate 96 (Ryoshu Diagnostics) plate to 1×10 4 cells/well, and measurement of viable cells was started using the xCELLigence real-time cell analyzer. . The next day, the plate was removed from the xCELLigence real-time cell analyzer, and 50 µL of each antibody prepared at each concentration (0.004, 0.04, 0.4, 4 µg/ml) was added to the plate. After reacting at room temperature for 15 minutes, 50 μL (2×10 4 cells/well) of the human PBMC solution prepared in (6-1) was added, and the measurement of viable cells was started by resetting the plate in the xCELLigence real-time cell analyzer. . The reaction was carried out under the conditions of 5% carbon dioxide gas and 37°C, and from the Cell Index value 72 hours after the addition of human PBMC, the cell proliferation inhibition rate (%) was calculated by the following formula. In addition, as for the Cell Index value used for calculation, the numerical value after normalizing so that the Cell Index value immediately before antibody addition might become 1 was used.

세포 증식 억제율(%)＝(A-B)×100/(A-1)Cell proliferation inhibition rate (%) = (A-B) × 100/(A-1)

A는 항체를 첨가하지 않은 웰에 있어서의 Cell Index값의 평균값(표적 세포와 인간 PBMC만), B는 각 웰에 있어서의 Cell Index값의 평균값을 나타낸다. A represents the average value of Cell Index values in wells to which no antibody was added (target cells and human PBMC only), and B represents the average value of Cell Index values in each well.

본 발명의 방법을 사용함으로써 환원 조건하에 있어서 종래 기술보다 높은 효율로 이중특이성 항체를 제작하는 것이 가능하다.
By using the method of the present invention, it is possible to produce bispecific antibodies with higher efficiency than the prior art under reducing conditions.

SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA <120> METHOD FOR PRODUCING POLYPEPTIDE HETEROMULTIMERS <130> C1-A1307P <150> JP 2013-200845 <151> 2013-09-27 <160> 81 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser 115 <210> 2 <211> 119 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 3 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 4 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 5 <211> 444 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 <210> 6 <211> 444 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 <210> 7 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 8 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 9 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 10 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 11 <211> 444 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 <210> 12 <211> 444 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Leu 435 440 <210> 13 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 15 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 15 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 16 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 16 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 17 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 18 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 19 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 19 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 20 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 20 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 21 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 22 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 23 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 23 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 24 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 24 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 25 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 26 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 27 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 27 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 28 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 29 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 30 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 31 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 32 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 33 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 34 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 35 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 36 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Glu Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 37 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Phe Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 38 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Tyr Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 39 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Thr Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 40 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Val Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 41 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Ile Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 42 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 43 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 43 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Glu Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 44 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Phe Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 45 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Tyr Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 46 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 46 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Thr Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 47 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 47 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Val Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 48 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Ile Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 49 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 49 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 50 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 50 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 51 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 51 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 52 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 52 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 53 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 54 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 54 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 55 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 55 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Lys Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 56 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 56 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Arg Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 57 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Asp Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 58 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 58 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Glu Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 59 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 59 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 60 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 61 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 61 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 145 150 155 160 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310 315 320 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Leu His His His His His His His His 435 440 445 <210> 62 <211> 455 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 62 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Cys Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Asp 435 440 445 Tyr Lys Asp Asp Asp Asp Lys 450 455 <210> 63 <211> 222 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 63 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 130 135 140 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 145 150 155 160 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 165 170 175 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 180 185 190 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 195 200 205 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 64 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 64 Asp Val Val Met Thr Gln Thr Pro Val Ser Met Ser Val Ser Leu Gly 1 5 10 15 Gly Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Val Ser Trp Tyr Ile Gln Lys Pro Ser Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Ile Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Asp Asp Leu Gly Val Tyr Tyr Cys Gly Gln Gly 85 90 95 Thr Gln Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 65 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 65 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro Lys Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Tyr Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 <210> 66 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 66 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro Lys Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 <210> 67 <211> 440 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 67 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Glu Ser Leu Ser Leu Ser Leu 435 440 <210> 68 <211> 439 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 68 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 115 120 125 Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val 130 135 140 Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser 145 150 155 160 Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu 165 170 175 Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 180 185 190 Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val 195 200 205 Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys 210 215 220 Thr Val Lys Glu Val Ser Lys Val Phe Ile Phe Pro Pro Lys Pro Lys 225 230 235 240 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 245 250 255 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 260 265 270 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 275 280 285 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 290 295 300 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 305 310 315 320 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 325 330 335 Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 340 345 350 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 355 360 365 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 370 375 380 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser 385 390 395 400 Glu Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 405 410 415 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 420 425 430 Leu Ser His Ser Pro Gly Lys 435 <210> 69 <211> 450 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 69 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg 225 230 235 240 Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Tyr Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro 450 <210> 70 <211> 450 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 70 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg 225 230 235 240 Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro 450 <210> 71 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 71 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Arg Gly Gly Pro 225 230 235 240 Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 <210> 72 <211> 446 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 72 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Lys Glu Val Ser Lys Val Phe 225 230 235 240 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Arg Thr Asp 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 73 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 73 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 74 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 74 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Lys Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 75 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 75 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Asp Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 76 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 76 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Arg Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 77 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 77 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Glu Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 78 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 78 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 79 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 79 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 80 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 80 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 81 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 81 Asp Val Val Met Thr Gln Thr Pro Val Ser Met Ser Val Ser Leu Gly 1 5 10 15 Gly Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Val Ser Trp Tyr Ile Gln Lys Pro Ser Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Ile Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Asp Asp Leu Gly Val Tyr Tyr Cys Gly Gln Gly 85 90 95 Thr Gln Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA <120> METHOD FOR PRODUCING POLYPEPTIDE HETERMULTIMERS <130> C1-A1307P <150> JP 2013-200845 <151> 2013-09-27 <160> 81 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser 115 <210> 2 <211> 119 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 3 <211> 447 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 4 <211> 447 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 5 <211> 444 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 <210> 6 <211> 444 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 <210> 7 <211> 447 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 8 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 9 <211> 447 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 10 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Ser Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 11 <211> 444 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 <210> 12 <211> 444 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Leu 435 440 <210> 13 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 15 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 15 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 16 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 16 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 17 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 18 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 19 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 19 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 20 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 20 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 21 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 22 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 23 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 23 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 24 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 24 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 25 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 26 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 27 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 27 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 28 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 29 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 30 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 31 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Lys Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 32 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 33 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 34 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 35 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 36 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Glu Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 37 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Phe Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 38 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Tyr Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 39 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Thr Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 40 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Val Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 41 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Ile Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 42 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 43 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 43 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Glu Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 44 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Phe Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 45 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Tyr Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 46 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 46 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Thr Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 47 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 47 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Val Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 48 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Ile Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 49 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 49 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 50 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 50 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 51 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 51 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Lys Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 52 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 52 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 53 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 54 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 54 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 55 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 55 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Lys Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 56 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 56 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Arg Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 57 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Asp Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 58 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 58 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Glu Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 59 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 59 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 60 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 61 <211> 447 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 61 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys 100 105 110 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys 115 120 125 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 145 150 155 160 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 165 170 175 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val 195 200 205 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 210 215 220 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310 315 320 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Leu His His His His His His His His 435 440 445 <210> 62 <211> 455 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 62 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Cys Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Asp 435 440 445 Tyr Lys Asp Asp Asp Asp Lys 450 455 <210> 63 <211> 222 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 63 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 130 135 140 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 145 150 155 160 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 165 170 175 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 180 185 190 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 195 200 205 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 64 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 64 Asp Val Val Met Thr Gln Thr Pro Val Ser Met Ser Val Ser Leu Gly 1 5 10 15 Gly Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Val Ser Trp Tyr Ile Gln Lys Pro Ser Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Ile Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Asp Asp Leu Gly Val Tyr Tyr Cys Gly Gln Gly 85 90 95 Thr Gln Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 65 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 65 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro Lys Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Tyr Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 <210> 66 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 66 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro Lys Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 <210> 67 <211> 440 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 67 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Arg Gly Gly Pro Lys Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Glu Ser Leu Ser Leu Ser Leu 435 440 <210> 68 <211> 439 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 68 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 115 120 125 Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val 130 135 140 Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser 145 150 155 160 Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu 165 170 175 Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 180 185 190 Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val 195 200 205 Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys 210 215 220 Thr Val Lys Glu Val Ser Lys Val Phe Ile Phe Pro Lys Pro Lys 225 230 235 240 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 245 250 255 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 260 265 270 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 275 280 285 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 290 295 300 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 305 310 315 320 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 325 330 335 Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu Gln Met Ala Lys 340 345 350 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 355 360 365 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 370 375 380 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser 385 390 395 400 Glu Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 405 410 415 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 420 425 430 Leu Ser His Ser Pro Gly Lys 435 <210> 69 <211> 450 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 69 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg 225 230 235 240 Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Tyr Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro 450 <210> 70 <211> 450 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 70 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg 225 230 235 240 Gly Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro 450 <210> 71 <211> 447 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 71 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Arg Gly Gly Pro 225 230 235 240 Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 <210> 72 <211> 446 <212> PRT <213> artificial sequence <220> <223> An artificially synthesized sequence <400> 72 Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 40 45 Ala Gln Ile Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Asn 65 70 75 80 Ile Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro 115 120 125 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 130 135 140 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 180 185 190 Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 195 200 205 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 210 215 220 Gly Cys Lys Pro Cys Ile Cys Thr Val Lys Glu Val Ser Lys Val Phe 225 230 235 240 Ile Phe Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 245 250 255 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 260 265 270 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 290 295 300 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 305 310 315 320 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 340 345 350 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 355 360 365 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 370 375 380 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Arg Thr Asp 385 390 395 400 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 405 410 415 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 420 425 430 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 73 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 73 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 74 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 74 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Lys Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 75 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 75 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Asp Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 76 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 76 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Arg Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 77 <211> 443 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 77 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205 Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 305 310 315 320 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Lys Glu 340 345 350 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 385 390 395 400 Phe Val Tyr Ser Glu Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 78 <211> 214 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 78 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 79 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 79 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 80 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 80 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 81 <211> 219 <212> PRT <213> Artificial sequence <220> <223> An artificially synthesized sequence <400> 81 Asp Val Val Met Thr Gln Thr Pro Val Ser Met Ser Val Ser Leu Gly 1 5 10 15 Gly Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 25 30 Asn Gly Asn Thr Tyr Val Ser Trp Tyr Ile Gln Lys Pro Ser Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Ile Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Asp Asp Leu Gly Val Tyr Tyr Cys Gly Gln Gly 85 90 95 Thr Gln Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125 Gln Leu Thr Ser Gly Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 145 150 155 160 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 180 185 190 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 195 200 205 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215

Claims (23)

a) 제1 항원 결합 활성을 갖고, Fc영역을 포함하는 제1 폴리펩티드의 호모체를 제공하는 단계,
b) 상기 제1 항원 결합 활성과 다른 제2 항원 결합 활성을 갖고, Fc영역을 포함하는 제2 폴리펩티드의 호모체를 제공하는 단계,
c) 힌지영역 내의 시스테인이 디설피드 결합의 이성화를 일으키는 것을 가능하게 하는 환원 조건하에서, 상기 제1 폴리펩티드의 호모체 및 상기 제2 폴리펩티드의 호모체를 함께 인큐베이트하는 단계, 및
d) 제1 및 제2 폴리펩티드를 포함하는 이종 다량체를 얻는 단계
를 포함하고,
상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서,
(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기
(2) EU 넘버링에 의한 357번 위치 및 370번 위치의 아미노산 잔기
(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기
에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동종의 전하를 가지며,
상기 제1 폴리펩티드의 CH3영역 및 상기 제2 폴리펩티드의 CH3영역 양쪽에 있어서, 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 같은 조의 아미노산 잔기가 각각 동종의 전하를 갖는 경우, 상기 제2 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기가 상기 제1 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기와는 반대의 전하를 갖고,
상기 제1 및 제2 폴리펩티드에 있어서 EU 넘버링에 의한 397번 위치의 아미노산이 Met(M), Phe(F) 또는 Tyr(Y)로 개변되어 있고,
상기 제1 및 제2 폴리펩티드의 Fc영역이 인간 IgG1 또는 IgG4 타입인
이종 다량체를 제조하기 위한 방법. a) having a first antigen-binding activity and providing a homo body of a first polypeptide comprising an Fc region;
b) having a second antigen-binding activity different from the first antigen-binding activity, providing a homo body of a second polypeptide comprising an Fc region;
c) incubating the homoform of the first polypeptide and the homoform of the second polypeptide together under reducing conditions that allow the cysteine in the hinge region to cause isomerization of the disulfide bond, and
d) obtaining a heteromultimer comprising the first and second polypeptides;
including,
In the CH3 region included in the Fc region of the first and/or second polypeptide,
(1) amino acid residues at positions 356 and 439 by EU numbering
(2) amino acid residues at positions 357 and 370 by EU numbering
(3) amino acid residues at positions 399 and 409 by EU numbering
1 to 3 sets of amino acid residues selected from the group of amino acid residues shown in
In both the CH3 region of the first polypeptide and the CH3 region of the second polypeptide, when the amino acid residues of the same group among the groups of amino acid residues shown in (1) to (3) have the same charge, the second The pair of amino acid residues in the CH3 region of the polypeptide has a charge opposite to that of the pair of amino acid residues in the CH3 region of the first polypeptide;
In the first and second polypeptides, the amino acid at position 397 by EU numbering is modified to Met (M), Phe (F) or Tyr (Y),
The Fc region of the first and second polypeptides is human IgG1 or IgG4 type
A method for preparing heteromultimers. 제1항에 있어서,
제1항의 단계 a)에 있어서 제1 폴리펩티드와 다량체를 형성하는 제3 폴리펩티드를 제공하는 단계를 포함하고, 또한 단계 b)에 있어서 제2 폴리펩티드와 다량체를 형성하는 제4 폴리펩티드를 제공하는 단계를 포함하는 제조방법. According to claim 1,
The method of claim 1 comprising the steps of providing a third polypeptide that multimers with the first polypeptide according to step a), and also providing a fourth polypeptide that multimers with the second polypeptide according to step b) A manufacturing method comprising a. 제1항 또는 제2항에 있어서,
상기 동종의 전하를 갖는 아미노산 잔기가 아래의 (A) 또는 (B) 중 어느 하나의 군에 포함되는 하나 이상의 아미노산 잔기로부터 선택되는 제조방법：
(A) 글루타민산(E), 아스파라긴산(D)；
(B) 리신(K), 아르기닌(R), 히스티딘(H). 3. The method of claim 1 or 2,
A production method wherein the amino acid residue having the same charge is selected from one or more amino acid residues included in any one of the following groups (A) or (B):
(A) glutamic acid (E), aspartic acid (D);
(B) Lysine (K), arginine (R), histidine (H). 제1항 또는 제2항에 있어서,
제1 및 제2 폴리펩티드에 있어서 각각 동종의 전하를 갖는 아미노산 잔기의 조가 아래의 (1)~(4) 중 어느 한 조의 아미노산 잔기의 조인 제조방법：
(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기
(2) EU 넘버링에 의한 357번 위치 및 370번 위치의 아미노산 잔기
(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기
(4) (i) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기, 및
(ii) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기. 3. The method of claim 1 or 2,
In the first and second polypeptides, the group of amino acid residues each having the same charge is a combination of amino acid residues of any one of the following (1) to (4):
(1) amino acid residues at positions 356 and 439 by EU numbering
(2) amino acid residues at positions 357 and 370 by EU numbering
(3) amino acid residues at positions 399 and 409 by EU numbering
(4) (i) the amino acid residues at positions 399 and 409 by EU numbering, and
(ii) amino acid residues at positions 356 and 439 by EU numbering. 제1항 또는 제2항에 있어서,
제1 및 제2 폴리펩티드에 있어서 각각 동종의 전하를 갖는 아미노산 잔기의 조가 아래의 조의 아미노산 잔기의 조인 제조방법：
(i) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기, 및
(ii) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기. 3. The method of claim 1 or 2,
A method for producing the first and second polypeptides, wherein a set of amino acid residues having the same charge, respectively, is a combination of the following set of amino acid residues:
(i) the amino acid residues at positions 399 and 409 by EU numbering, and
(ii) amino acid residues at positions 356 and 439 by EU numbering. 제1항 또는 제2항에 있어서,
제1 및/또는 제2 폴리펩티드에 있어서 제1 및/또는 제2 폴리펩티드의 CH3영역의 안정성이 불안정해지도록 아미노산이 개변되어 있는 제조방법. 3. The method of claim 1 or 2,
In the first and/or second polypeptide, the amino acid is modified so that the stability of the CH3 region of the first and/or second polypeptide is unstable. 삭제delete 삭제delete 삭제delete 제1항에 있어서,
상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서,
(1) EU 넘버링에 의한 356번 위치 및 439번 위치의 아미노산 잔기
(2) EU 넘버링에 의한 360번 위치 및 371번 위치의 아미노산 잔기
(3) EU 넘버링에 의한 399번 위치 및 409번 위치의 아미노산 잔기
에 나타내는 아미노산 잔기의 조로부터 선택되는 1조 내지 3조의 아미노산 잔기가 동종의 전하를 갖고,
상기 제1 폴리펩티드의 CH3영역 및 상기 제2 폴리펩티드의 CH3영역 양쪽에 있어서, 상기 (1)~(3)에 나타내는 아미노산 잔기의 조 중 같은 조의 아미노산 잔기가 각각 동종의 전하를 갖는 경우, 상기 제2 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기가 상기 제1 폴리펩티드의 CH3영역에 있어서의 당해 조의 아미노산 잔기와는 반대의 전하를 갖는
이종 다량체를 제조하기 위한 방법. According to claim 1,
In the CH3 region included in the Fc region of the first and/or second polypeptide,
(1) amino acid residues at positions 356 and 439 by EU numbering
(2) amino acid residues at positions 360 and 371 by EU numbering
(3) amino acid residues at positions 399 and 409 by EU numbering
1 to 3 sets of amino acid residues selected from the group of amino acid residues shown in
In both the CH3 region of the first polypeptide and the CH3 region of the second polypeptide, when the amino acid residues of the same group among the groups of amino acid residues shown in (1) to (3) have the same charge, the second The pair of amino acid residues in the CH3 region of the polypeptide has a charge opposite to that of the pair of amino acid residues in the CH3 region of the first polypeptide
A method for preparing heteromultimers. 제1항에 있어서,
상기 제1 및/또는 제2 폴리펩티드의 Fc영역에 포함되는 CH3영역에 있어서의 EU 넘버링에 의한 392번 위치의 아미노산이 개변되어 있는 제조방법.According to claim 1,
A production method in which the amino acid at position 392 by EU numbering in the CH3 region included in the Fc region of the first and/or second polypeptide is altered. 제1항 또는 제11항에 있어서,
제1 및/또는 제2 폴리펩티드에 있어서
EU 넘버링에 의한 392번 위치가 Asp(D), Glu(E), Thr(T), Val(V) 또는 Ile(I)로 개변되어 있는
제조방법. 12. The method of claim 1 or 11,
for the first and/or second polypeptide
Position 392 by EU numbering is modified with Asp (D), Glu (E), Thr (T), Val (V) or Ile (I)
manufacturing method. 제1항 또는 제11항에 있어서,
제1 및 제2 폴리펩티드에 있어서 EU 넘버링에 의한 397번 위치가 Phe(F) 또는 Tyr(Y)로 개변되어 있는 제조방법. 12. The method of claim 1 or 11,
In the first and second polypeptides, position 397 by EU numbering is modified with Phe (F) or Tyr (Y). 제1항 또는 제11항에 있어서,
제1 폴리펩티드에 있어서 EU 넘버링에 의한 356번 위치가 Lys(K)로 개변되어 있고, 또한 EU 넘버링에 의한 397번 위치가 Phe(F) 또는 Tyr(Y)로 개변되어 있으며, 제2 폴리펩티드에 있어서 EU 넘버링에 의한 397번 위치가 Phe(F) 또는 Tyr(Y)로 개변되어 있고, 또한 EU 넘버링에 의한 439번 위치가 Glu(E)로 개변되어 있는 제조방법. 12. The method of claim 1 or 11,
In the first polypeptide, position 356 by EU numbering is modified with Lys (K), and position 397 by EU numbering is modified with Phe (F) or Tyr (Y), in the second polypeptide A manufacturing method in which position 397 by EU numbering is modified with Phe (F) or Tyr (Y), and position 439 by EU numbering is modified with Glu (E). 제1항 또는 제11항에 있어서,
상기 단계 a) 및 b)가 제1 폴리펩티드의 호모체를 생산하는 세포주와 제2 폴리펩티드의 호모체를 생산하는 세포주를 혼합함으로써 실시되고, 상기 단계 c)가 당해 배양상청 중에서 실시되는 제조방법. 12. The method of claim 1 or 11,
A method wherein steps a) and b) are carried out by mixing a cell line producing a homo body of the first polypeptide with a cell line producing a homo body of the second polypeptide, and step c) is carried out in the culture supernatant. 제1항 또는 제11항에 있어서,
상기 이종 다량체가 다중특이성 항체 또는 헤테로 Fc 융합 단백질인 제조방법. 12. The method of claim 1 or 11,
A method for producing the heteromultimer is a multispecific antibody or hetero Fc fusion protein. 제1항 또는 제11항에 있어서,
상기 이종 다량체가 이중특이성 항체인 제조방법. 12. The method of claim 1 or 11,
The method for producing the heteromultimer is a bispecific antibody. 제1항 또는 제11항에 있어서,
단계 c)가 환원제와의 접촉을 포함하는 제조방법. 12. The method of claim 1 or 11,
A process wherein step c) comprises contacting with a reducing agent. 제18항에 있어서,
단계 c)가 글루타티온, L-시스테인, 디티오트레이톨, β-메르캅토-에탄올, TCEP 및 2-MEA로 이루어진 군으로부터 선택되는 작용물질의 첨가를 포함하는 제조방법. 19. The method of claim 18,
A process wherein step c) comprises the addition of an agent selected from the group consisting of glutathione, L-cysteine, dithiothreitol, β-mercapto-ethanol, TCEP and 2-MEA. 제19항에 있어서,
단계 c)가 글루타티온, 2-MEA로부터 선택되는 작용물질의 첨가를 포함하는 제조방법. 20. The method of claim 19,
A process wherein step c) comprises the addition of an agonist selected from glutathione, 2-MEA. 삭제delete 삭제delete 삭제delete
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